U.S. patent application number 11/125577 was filed with the patent office on 2005-09-22 for method of isolating bile duct progenitor cells.
This patent application is currently assigned to ES Cell International Pte Ltd.. Invention is credited to Homa, Monica, Pang, Kevin.
Application Number | 20050208653 11/125577 |
Document ID | / |
Family ID | 23898374 |
Filed Date | 2005-09-22 |
United States Patent
Application |
20050208653 |
Kind Code |
A1 |
Pang, Kevin ; et
al. |
September 22, 2005 |
Method of isolating bile duct progenitor cells
Abstract
The present invention relates to a substantially pure population
of viable bile duct progenitor cells, and methods for isolating
such cells. The present invention further concerns certain
therapeutic uses for such progenitor cells, and their progeny.
Inventors: |
Pang, Kevin; (Belmont,
MA) ; Homa, Monica; (Marblehead, MA) |
Correspondence
Address: |
FISH & NEAVE IP GROUP
ROPES & GRAY LLP
ONE INTERNATIONAL PLACE
BOSTON
MA
02110-2624
US
|
Assignee: |
ES Cell International Pte
Ltd.
Singapore
SG
|
Family ID: |
23898374 |
Appl. No.: |
11/125577 |
Filed: |
May 10, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11125577 |
May 10, 2005 |
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08973938 |
Aug 21, 1998 |
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08973938 |
Aug 21, 1998 |
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PCT/US96/09656 |
Jun 7, 1996 |
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08973938 |
Aug 21, 1998 |
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08478064 |
Jun 7, 1995 |
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5861313 |
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Current U.S.
Class: |
435/366 ;
435/325 |
Current CPC
Class: |
A61P 31/20 20180101;
A61P 31/04 20180101; C12N 2501/113 20130101; A61K 35/413 20130101;
C12N 2501/115 20130101; A61K 35/39 20130101; C12N 2501/11 20130101;
C12N 2501/148 20130101; C12N 5/0678 20130101; A61P 1/16 20180101;
A61K 35/407 20130101; C12N 5/0672 20130101 |
Class at
Publication: |
435/366 ;
435/325 |
International
Class: |
C12N 005/08; C12N
005/06 |
Claims
1. (canceled)
2. A cellular composition comprising, as the cellular component, a
substantially pure population of viable pancreatic progenitor
cells, which progenitor cells are capable of proliferation in a
culture medium and differentiate to pancreatic lineages.
3. The composition of claim 2, wherein at least 80% of the viable
cells in the composition are progenitor cells.
4. The composition of claim 2, which progenitor cells are from a
mammal.
5. The composition of claim 4, which mammal is a transgenic
mammal.
6. The composition of claim 4, which mammal is a primate.
7. The composition of claim 6, which mammal is a human.
8. The composition of claim 4, which mammal is a miniature
swine.
9-13. (canceled)
14. The composition of claim 2, wherein the progenitor cells
express one or more of STF-1; a PAX gene; PTF-1; hXBP-1; an HNF
gene; villin; tyrosine hydroxylase; insulin; glucagon; or
neuropeptide Y.
15. The composition of claim 14, wherein the progenitor cells
express STF-1 and PAX6.
16-19. (canceled)
20. The composition of claim 2, which progenitor cells can be
maintained in culture for at least about 7 days.
21. (canceled)
22. A cellular composition consisting essentially of, as the
cellular population, viable pancreatic progenitor cells capable of
self-regeneration in a culture medium, which progenitor cells
differentiate to members of the pancreatic lineage.
23. The composition of claim 22, which progenitor cells are
isolated from a hepatic duct tissue, or are the progeny
thereof.
24. The composition of claim 22, which progenitor cells are
isolated from cystic duct tissue, or are the progeny thereof.
25. The composition of claim 22, which progenitor cells are
isolated from pancreatic duct tissue, or are the progeny
thereof.
26. The composition of claim 22, which progenitor cells are
isolated from common bile duct tissue, or are the progeny
thereof.
27. The composition of claim 22, which progenitor cells are
responsive to one or more growth factor selected from a group
consisting of IGF, EGF, TGF, FGF, HGF and VEGF, or orthologous or
paralogous factors thereof.
28. A cellular composition comprising pancreatic progenitor cells,
wherein the progenitor cells are at least 80% pure, are capable of
self-regeneration in a culture medium and differentiate to
pancreatic lineages.
29. The composition of claim 28, which progenitor cells are
inducible to differentiate into pancreatic islet cells.
30. The composition of claim 29, which islet cells are pancreatic
.beta. islet cells.
31. The composition of claim 29, which islet cells are pancreatic
.alpha. islet cells.
32. The composition of claim 29, which islet cells are pancreatic
.delta. islet cells.
33. The composition of claim 29, which islet cells are pancreatic
.phi. islet cells.
34. The composition of claim 28, wherein the progenitor cells are
characterized by expression of STF-1 and PAX6.
37. A pharmaceutical composition comprising the cellular
composition of claim 2.
39. A pharmaceutical composition comprising the cellular
composition of claim 22.
40. A pharmaceutical composition comprising the cellular
composition of claim 28.
41-54. (canceled)
55. A method for treating a disorder characterized by insufficient
insulin activity, in a subject, comprising introducing into the
subject a pharmaceutical composition including a substantially pure
population of pancreatic progenitor cells and a pharmaceutically
acceptable carrier.
56. The method of claim 55, wherein the subject is a human.
57. The method of claim 55, wherein the disorder is an insulin
dependent diabetes.
58. The method of claim 57, wherein the insulin dependent diabetes
is type I diabetes.
59. The method of claim 55, wherein the pancreatic progenitor cells
are induced to differentiate into pancreatic islet cells in the
subject.
60. The method of claim 55, wherein the pancreatic progenitor cells
are induced to differentiate into pancreatic .beta. islet cells in
culture prior to introduction into the subject.
61. The method of claim 55, wherein the pancreatic progenitor cells
are characterized by expression of STF-1 and PAX6.
62. A method for treating a disorder characterized by insufficient
liver function, in a subject, comprising introducing into the
subject a pharmaceutical composition including hepatic progenitor
cells and a pharmaceutically acceptable carrier.
63. The method of claim 62, wherein the subject is a human.
64. The method of claim 62, wherein the disorder is selected from
the group consisting of cirrhosis, hepatitis B, hepatitis C, sepsis
or ELAD.
65. The method of claim 62, wherein the hepatic progenitor cells
are induced to differentiate into hepatocytes in the subject.
66. The method of claim 62, wherein the hepatic progenitor cells
are induced to differentiate into hepatocytes in culture prior to
introduction into the subject.
67. The method of claim 62, wherein the hepatic progenitor cells
express HNF3, alphafetoprotein (AFP), albumin, and ATBF-1, and
differentiate into hepatocytes.
Description
BACKGROUND OF THE INVENTION
[0001] During the early stages of embryogenesis cells are
totipotent and are capable of multidirectional differentiation. As
development proceeds, the totipotent cells become determined and
committed to differentiate into a given specialized cell type.
Final differentiation is associated with the acquisition of
specialized cell functions. Thus, the differentiated somatic cells
maintain their specialized features throughout the life span of the
organism, probably through sustained interactions between the
genome and its microenvironment and cell-cell interactions
(DiBerardino et al. 1984, Science 224: 946-952; Wetts and Fraser,
1988, Science 239: 1142-1144; Fisher, 1984, PNAS 81:
4414-4418).
[0002] Because of the tremendous potential of progenitor cells to
differentiate into distinct lineages, there has always existed a
need for a continuous source of these isolated pluripotent
progenitor cells. The pluripotent progenitor cells could be
extremely useful in the treatment of different disorders that are
characterized by insufficient or abnormal functioning of the fully
differentiated cells in a given organ, as for example in the human
pancreas or liver.
SUMMARY OF THE INVENTION
[0003] The present invention relates to substantially pure
preparations of viable progenitor cells and methods for isolating
such cells. The present invention further concerns certain uses for
such progenitor cells and their progeny.
[0004] In general, the invention features a cellular composition
including, as the cellular component, a substantially pure
population of viable bile duct progenitor cells which progenitor
cells are capable of proliferation in a culture medium. In a
preferred embodiment, the cellular composition has fewer than about
20%, more preferably fewer than about 10%, most preferably fewer
than about 5% of lineage committed cells.
[0005] In general, the progenitor cells of the present invention
are proliferative cells which can differentiate into cells making
up the tissues of the gut, e.g., the liver, pancreas, gallbladder,
intestines, etc. That is, the progenitor cells can give rise to
differentiated cells of hepatic, pancreatic, gallbladder or
intestinal lineages. However, as described in the pending examples,
the subject method can be used to isolate populations of
hematopoietic stem/progenitor cells. In preferred embodiments, the
subject progenitor cells are pluripotent, e.g., the progenitor
cells are capable of differentiating into two or more distinct
lineages.
[0006] In one embodiment, the progenitor cells of the present
invention are characterized by an ability for self-regeneration in
a culture medium and differentiation to pancreatic lineages. In a
preferred embodiment, the progenitor cells are inducible to
differentiate into pancreatic islet cells, e.g., .beta. islet
cells, .alpha. islet cells, .delta. islet cells, or .phi. islet
cells. Such pancreatic progenitor cells may be characterized in
certain circumstances by the expression of one or more of:
homeodomain type transcription factors such as STF-1; PAX gene(s)
such as PAX6; PTF-1; hXBP-1; HNF genes(s); villin; tyrosine
hydroxylase; insulin; glucagon; and/or neuropeptide Y.
[0007] In another embodiment, the invention features progenitor
cells, such as may be obtained from a non-hepatic bile duct
according to the present invention, which are characterized by an
ability to differentiate (e.g., by induction) into hepatocytes when
maintained in culture. Exemplary hepatic progenitor cells may be
characterized by the expression of one or more of: a hepatocyte
nuclear factor (HNF) transcription factor, e.g., HNF1.alpha.,
HNF1.beta., HNF3.beta., HNF3.gamma., and/or HNF4; ATBF1; AFP; a LIM
type homeobox gene such as Islet-1; a "forkhead" transcription
factor, such as fkh-1; a CCAAT-enhancer binding protein (C/EBP),
such as C/EBP-.beta.; oval cell marker OV-6; cytokeratins (such as
type 7 or 19), mucin Span-1; OC.2; OC.3; and/or .gamma.-glutamyl
transpeptidase.
[0008] In still another embodiment, the invention features
hematopoietic progenitor cells which may differentiate into one or
more of an erythrocyte, a megakaryocyte, a monocyte, a granulocyte
and/or an eosinophils as well as fully differentiated lymphoid
cells such as B lymphocytes and T lymphocytes. Certain of the
subject hematopoietic progenitor cells may be characterized by
expression of such markers as c-kit, CD34 and/or CD33, as well as
OV-6. CAM5.2 and/or cytokeratin type 7 or 18.
[0009] In yet another embodiment, the invention features a
pharmaceutical composition including as the cellular component a
substantially pure population of viable bile duct progenitor cells,
which progenitor cells are capable of proliferation in a culture
medium.
[0010] In general, the preferred progenitor cells will be of
mammalian origin, e.g., cells isolated from a primate such as a
human, from a miniature swine, or from a transgenic mammal, or are
the cell culture progeny of such cells.
[0011] In preferred embodiments, the subject progenitor cells can
be maintained in cell/tissue culture for at least about 7 days,
more preferably for at least about 14 days, most preferably for at
least about 21 days or longer.
[0012] In another aspect, the invention features a cellular
composition comprising, as a cellular population, at least 75%
(though more preferably at least 80, 90 or 95%) progenitor cells
isolated from a bile duct and capable of self-regeneration in a
culture medium.
[0013] In yet another aspect, the invention features, a cellular
composition consisting essentially of, as the cellular population,
viable non-hepatic duct progenitor cells capable of
self-regeneration in a culture medium and differentiation to
members of the hepatic, pancreatic and gallbladder lineages. For
instance, in certain embodiments the progenitor cells are isolated
from cystic duct explants, pancreatic duct explants common bile
duct explants, or are the cell culture progeny of such cells.
[0014] Another aspect of the invention features a method for
isolating progenitor cells from a bile duct. In general, the method
provides for culturing an isolated population of cells having a
microarchitecture of a mammalian bile duct, e.g. a micro-organ
explant in which the original epithelial-mesenchymal
microarchitecture is maintained, wherein the dimensions of the
explant provide the isolated population of cells as maintainable in
culture for at least twenty-four hours, and includes in the
population of cells at least one progenitor cell which can
proliferate under such culture conditions. The cultured cell
population is contacted with an agent, e.g., a mitogenic agent such
as a growth factor which agent causes proliferation of progenitor
cells in the cultured population. Subsequently, progenitor cells
from the explant that proliferate in response to the agent are
isolated, such as by direct mechanical separation of newly emerging
buds from the rest of the explant or by dissolution of all or a
portion of the explant and subsequent isolation of the progenitor
cell population.
[0015] In another preferred embodiment, the agent is a growth
factor. e.g., the growth factor is selected from a group consisting
of IGF, TGF, FGF, EGF, HGF, or VEGF. In other embodiments, the
growth factor is a member of the TGF.beta. superfamily, preferably
of the DVR (dpp and vgl related) family, e.g., BMP2 and/or
BMP7.
[0016] In another preferred embodiment, the population of cells is
cultured in a medium deficient in biological extracts. e.g.,
deficient in serum.
[0017] In a preferred embodiment, the bile duct is a common bile
duct.
[0018] In another aspect, the invention features, a method for
screening a compound for ability to modulate one of growth,
proliferation, and/or differentiation of progenitor cells obtained
from a bile duct, including: (i) establishing an isolated
population of cells having a microarchitecture of a mammalian bile
duct, e.g., a micro-organ explant in which the original
epithelial-mesenchymal microarchitecture is maintained, wherein the
dimensions of the explant provide the isolated population of cells
as maintainable in culture for at least twenty-four hours, and
includes at least one progenitor cell which has the ability to
proliferate in the culture; (ii) contacting the population of cells
with a test compound; and (iii) detecting one of growth,
proliferation, and/or differentiation of the progenitor cells in
the population, wherein a statistically significant change in the
extent of one of growth, proliferation, and/or differentiation in
the presence of the test compound relative to the extent of one of
growth, proliferation, and/or differentiation in the absence of the
test compound indicates the ability of the test compound to
modulate one of the growth, proliferation, and/or
differentiation.
[0019] In another aspect the invention features, a method for
treating a disorder characterized by insufficient insulin activity,
in a subject including introducing into the subject a
pharmaceutical composition including pancreatic progenitor cells,
or differentiated cells arising therefrom and a pharmaceutically
acceptable carrier.
[0020] In a preferred embodiment the subject is a mammal, e.g., a
primate, e.g., a human.
[0021] In another preferred embodiment the disorder is an insulin
dependent diabetes, e.g., type I diabetes.
[0022] In yet another preferred embodiment, the pancreatic
progenitor cells are induced to differentiate into pancreatic islet
cells, e.g., .beta. islet cells, .alpha. islet cells, .delta. islet
cells, or .phi. islet cells, subsequent to being introduced into
the subject. Preferably, the pancreatic progenitors cells are
induced to differentiate into pancreatic islet, e.g., .beta. islet
cells, .alpha. islet cells, .delta. islet cells or .phi. islet
cells, in culture prior to introduction into the subject.
[0023] In another aspect, the invention features, a method for
treating a disorder characterized by insufficient liver function,
in a subject comprising introducing into the subject a
pharmaceutical composition including hepatic progenitor cells and a
pharmaceutically acceptable carrier.
[0024] In a preferred embodiment, the subject is a mammal, e.g., a
primate, e.g., a human.
[0025] In another preferred embodiment the disorder is selected
from the group consisting of cirrhosis, hepatitis B, hepatitis C,
sepsis, or ELAD. In yet another preferred embodiment, the hepatic
progenitor cells are induced to differentiate into hepatocytes
subsequent to being introduced into the subject. Preferably, the
hepatic progenitors cells are induced to differentiate into
hepatocytes in culture prior to introduction into the subject.
[0026] The practice of the present invention will employ, unless
otherwise indicated, conventional techniques of cell biology, cell
culture, molecular biology, transgenic biology, microbiology,
recombinant DNA, and immunology, which are within the skill of the
art. Such techniques are described in the literature. See, for
example, Molecular Cloning A Laboratory Manual; 2nd Ed., ed. by
Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory
Press: 1989); DNA Cloning, Volumes I and II (D. N. Glover ed.,
1985); Oligonucleotide Synthesis (M. J. Gait ed., 1984); Mullis et
al. U.S. Pat. No. 4,683,195; Nucleic Acid Hybridization (B. D.
Hames & S. J. Higgins eds. 1984); Transcription And Translation
(B. D. Hames & S. J. Higgins eds. 1984); Culture Of Animal
Cells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells
And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To
Molecular Cloning (1984); the treatise, Methods In Enzymology
(Academic Press. Inc., N.Y.); Gene Transfer Vectors For Mammalian
Cells (J. H. Miller and M. P. Calos eds. 1987, Cold Spring Harbor
Laboratory); Methods In Enzymology, Vols. 154 and 155 (Wu et al.
eds.), Immunochemical Methods In Cell And Molecular Biology (Mayer
and Walker, eds. Academic Press, London, 1987); Handbook Of
Experimental Immunology, Volumes I-IV (D. M. Weir and C. C.
Blackwell, eds. 1986); Manipulating the Mouse Embryo, (Cold Spring
Harbor Laboratory Press. Cold Spring Harbor. N.Y. 1986).
[0027] Other features and advantages of the invention will be
apparent from the following detailed description, and from the
claims.
DETAILED DESCRIPTION OF THE DRAWINGS
[0028] FIG. 1 is a graph depicting average number of BrdU positive
nuclei in a common bile duct explant, 24 hours after administration
of a growth factor. Growth factors, EGF, TGF-.alpha.and basic FGF
(bFGF) were administered in three doses: 1 ng/ml, 10 ng/ml and 100
ng/ml. DMEM minimal media was used as a control. Average
fluorescence intensity was determined based on the number of
positive nuclei.
[0029] FIG. 2 is a graph depicting the percentage of BrdU labeled
positive cells in a common bile duct explant, 24 hours after
administration of a growth factor. Growth factors. EGF,
TGF-.alpha., and bFGF were administered and DMEM minimal media was
used as a control.
[0030] FIG. 3 is a micrographs depicting in vitro growth and
expansion of the cells within a common bile duct explant in
response to administration of growth factor bFGF.
[0031] FIGS. 4A-4C a micrographs (120.times.) illustrating the
types of cells expanded from bile duct microorgan cultures by IGF-1
stimulation. The addition of IGF-1 at 100 ng/ml resulted in at
least three types of responses; no growth or change in morphology
(FIG. 4A); the appearance of "spiny" colonies which are termed
"N-type" colonies due to the neurite-like appearance of the colony
extensions (FIG. 4B); and the appearance of "L-type" colonies for
liver-like, due to the epithelial blebs and formation of red blood
cell foci (FIG. 4C) which were identical in appearance to cultured
embryonic liver (not shown).
[0032] FIG. 5 is a graph demonstrating that ductal microorgan
cultures on matrigel can give riase to multiple colony types.
DETAILED DESCRIPTION OF THE INVENTION
[0033] The ability to isolate distinct populations of progenitor
cells has been an important problem in modern biology. It can be
easily envisioned that such isolated pluripotent progenitor cells
could be very useful for treatment of various disorders associated
with loss or abnormal functioning of fully differentiated cells in
a given organ. For example, the ability to introduce isolated
progenitor cells capable of subsequent differentiation, either in
culture or when introduced into a subject, into functional islet
cells, would have important implications for the treatment of
insulin-dependent diabetes. In the same manner, the ability to
deliver purified hepatic progenitor cells, having the ability to
differentiate into mature hepatocytes, could be potentially useful
in the process of liver regeneration or for treatment of disorders
characterized by insufficient liver function. Prior to the instant
invention, there was no apparently reliable source of purified
populations of progenitor cells from gut tissue capable of further
differentiation into distinct pancreatic, hepatic, gallbladder,
intestinal or hematopoietic lineages, either in culture or when
introduced into a subject.
[0034] Accordingly, certain aspects of the present invention relate
to isolated populations of progenitor cells capable of subsequent
differentiation to distinct pancreatic, hepatic, gallbladder,
intestinal or hematopoietic lineages, methods for isolating such
cells and therapeutic uses for such cells.
[0035] For convenience, certain terms employed in the
specification, examples, and appended claims are collected
here.
[0036] The term "culture medium" is recognized in the art, and
refers generally to any substance or preparation used for the
cultivation of living cells. Accordingly, a "tissue culture" refers
to the maintenance or growth of tissue, e.g., explants of organ
primordia or of an adult organ in vitro so as to preserve its
architecture and function. A "cell culture" refers to a growth of
cells in vitro; although the cells proliferate they do not organize
into tissue per se.
[0037] Tissue and cell culture preparations of the subject
micro-organ explants and amplified progenitor cell populations can
take on a variety of formats. For instance, a "suspension culture"
refers to a culture in which cells multiply while suspended in a
suitable medium. Likewise, a "continuous flow culture" refers to
the the cultivation of cells or ductal explants in a continuous
flow of fresh medium to maintain cell growth. e.g. viablity. The
term "conditioned media" refers to the supernatant, e.g. free of
the cultured cells/tissue, resulting after a period of time in
contact with the cultured cells such that the media has been
altered to include certain paracrine and/or autocrine factors
produced by the cells and secreted into the culture.
[0038] The terms "explant" and "micro-organ explant" refer to a
portion of an organ taken from the body and grown in an artificial
medium.
[0039] The term "tissue" refers to a group or layer of similarly
specialized cells which together perform certain special
functions.
[0040] The term "organ" refers to two or more adjacent layers of
tissue which layers of tissue maintain some form of cell-cell
and/or cell-matrix interaction to form a microarchitecture.
[0041] The term "lineage committed cell" refers to a progenitor
cell that is no longer pluripotent but has been induce to
differentiate into a specific cell type, e.g., a pancreatic,
hepatic or intestinal cell.
[0042] The term "progenitor cell" refers to an undifferentiated
cell which is capable of proliferation and giving rise to more
progenitor cells having the ability to generate a large number of
mother cells that can in turn give rise to differentiated, or
differentiable daughter cells. As used herein, the term "progenitor
cell" is also intended to encompass a cell which is sometimes
referred to in the art as a "stem cell". In a preferred embodiment,
the term "progenitor cell" refers to a generalized mother cell
whose descendants (progeny) specialize, often in different
directions, by differentiation, e.g., by acquiring completely
individual characters, as occurs in progressive diversification of
embryonic cells and tissues. A "bile duct progenitor cells" refers
to progenitor cells arising in tissue of a bile duct and giving
rise to such differentiated progeny as, for example, hepatic,
pancreatic, intestinal, gallbladder or hematopoietic lineages.
[0043] As used herein the term "bile duct" refers to an intricate
system of ducts, e.g., generally tubular structures used for
secretion, either neonatal or adult. The term includes the hepatic
duct, cystic duct, and pancreatic duct. The term "pancreatic duct"
includes the accessory pancreatic duct, dorsal pancreatic duct,
main pancreatic duct and ventral pancreatic duct. The term bile
duct also encompasses the common bile duct. The main function of
the bile duct is to allow bile and other materials to drain from
these organs and enter the gastrointestinal tract.
[0044] As used herein the term "common bile duct" refers to a
region of the bile duct either adult or neonatal, originating from
the liver bile canaliculi and extending down to the papilla of
Vater at the duodenal junction. The common bile duct is continuous
with hepatic, cystic and certain pancreatic ducts.
[0045] As used herein the term "non-hepatic bile duct" refers to
that bile duct tissue which is not hepatic duct tissue, e.g., those
portions of the common bile duct posterior to the hepatic duct. In
addition, the term includes cystic and pancreatic ducts.
[0046] As used herein the term "substantially pure", with respect
to progenitor cells, refers to a population of progenitor cells
that is at least about 75%, preferably at least about 85%, more
preferably at least about 90% and most preferably at least about
95% pure with respect to progenitor cells making up a total cell
population. Recast, the term "substantially pure" refers to a
population of progenitor cell of the present invention than contain
fewer than about 20%, more preferably fewer than about 10%, most
preferably fewer than about 5%, of lineage committed cells in the
original unamplified and isolated population prior to subsequent
culturing and amplification.
[0047] As used herein the term "cellular composition" refers to a
preparation of cells, which preparation may include, in addition to
the cells non-cellular components such as cell culture media, e.g.
proteins, amino acids, nucleic acids, nucleotides, co-enzyme,
anti-oxidants, metals and the like. Furthermore, the cellular
composition can have components which do not affect the growth or
viability of the cellular component, but which are used to provide
the cells in a particular format, e.g., as polymeric matrix for
encapsulation or a pharmaceutical preparation.
[0048] As used herein the term "animal" refers to mammals,
preferably mammals such as humans. Likewise, a "patient" or
"subject" to be treated by the method of the invention can mean
either a human or non-human animal.
[0049] As described below, in a preferred embodiment, the
progenitor cells of the present invention are pancreatic or hepatic
progenitor cells. The term "pancreas" is art recognized, and refers
generally to a large, elongated racemose gland situated
transversely behind the stomach, between the spleen and duodenum.
The pancreatic exocrine function, e.g., external secretion,
provides a source of digestive enzymes. Indeed, "pancreatin" refers
to a substance from the pancreas containing enzymes principally
amylase, protease, and lipase, which substance is used as a
digestive aid. The exocrine portion is composed of several serous
cells surrounding a lumen. These cells synthesize and secrete
digestive enzymes such as trypsinogen, chymotrypsinogen,
carboxypeptidase, ribonuclease, deoxyribonuclease, triacylglycerol
lipase phospholipase A.sub.2, elastase, and amylase.
[0050] The endocrine portion of the pancreas is composed of the
islets of Langerhans. The islets of Langerhans appear as rounded
clusters of cells embedded within the exocrine pancreas. Four
different types of cells--.alpha., .beta., .delta., and .phi.--have
been identified in the islets. The .alpha. cells constitute about
20% of the cells found in pancreatic islets and produce the hormone
glucagon. Glucagon acts on several tissues to make energy available
in the intervals between feeding. In the liver, glucagon causes
breakdown of glycogen and promotes gluconeogenesis from amino acid
precursors. The .delta. cells produce somatostatin which acts in
the pancreas to inhibit glucagon release and to decrease pancreatic
exocrine secretion. The hormone pancreatic polypeptide is produced
in the .phi. cells. This hormone inhibits pancreatic exocrine
secretion of bicarbonate and enzymes, causes relaxation of the
gallbladder, and decreases bile secretion. The most abundant cell
in the islets, constituting 60-80% of the cells, is the .beta.
cell, which produces insulin. Insulin is known to cause the storage
of excess nutrients arising during and shortly after feeding. The
major target organs for insulin are the liver, muscle, and
fat-organs specialized for storage of energy.
[0051] The term "pancreatic progenitor cell" refers to a cell which
can differentiate into a cell of pancreatic lineage, e.g. a cell
which can produce a hormone or enzyme normally produced by a
pancreatic cell. For instance, a pancreatic progenitor cell may be
caused to differentiate, at least partially, into .alpha., .beta.,
.delta., or .phi. islet cell or a cell of exocrine fate. The
pancreatic progenitor cells of the invention can also be cultured
prior to administration to a subject under conditions which promote
cell proliferation and differentiation. These conditions include
culturing the cells to allow proliferation and confluence in vitro
at which time the cells can be made to form pseudo islet-like
aggregates or clusters and secrete insulin, glucagon, and
somatostatin.
[0052] The term "liver" refers to the large, dark-red gland in the
upper part of the abdomen on the right side, just beneath the
diaphragm. Its manifold functions include storage and filtration of
blood, secretion of bile, conversion of sugars into glycogen, and
many other metabolic activities.
[0053] The liver is a gland that supplies bile to intestine. In
adult vertebrates, this function is a minor one, but the liver
originally arose as a digestive gland in lower chordates.
Throughout the liver, a network of tiny tubules collects bile--a
solution of salts, bilirubin (made when hemoglobin from red blood
cells is broken down in liver) and fatty acids. Bile accumulates in
the gall bladder, which empties into the small intestine by way of
a duct. Bile has two functions in the intestine. First, it acts as
a detergent, breaking fat into small globules that can be attacked
by digestive enzymes. Second, and more important, bile salts aid in
the absorption of lipids form the intestine; removal of the gall
bladder sometimes causes difficulty with lipid absorption.
[0054] Digested food molecules absorbed into the bloodstream from
the intestine pass directly to the liver by way of the hepatic
portal vein. Before these molecules pass on into the rest of the
body, the liver may change their concentration and even their
chemical structure. The liver performs a vital role in detoxifying
otherwise poisonous substances. In addition, it stores food
molecules that reach it form the intestine, converts them
biochemically, and releases them back into the blood at a
controlled rate. For instance, the liver removes glucose from the
blood under the influence of the hormone insulin and stores it as
glycogen. When the level of glucose in the blood falls, the hormone
glucagon causes the liver to break down glycogen and release
glucose into the blood.
[0055] The liver also synthesizes many of the blood proteins (e.g.,
albumins) and releases them into the blood when they are needed. In
addition, the liver converts nitrogenous wastes into the form of
urea for excretion by the kidneys. With the kidneys, the liver is
vital in regulating what the blood contains when it reaches all the
other organs of the body. Because the liver is the body's major
organ for making all these biochemical adjustments severe liver
damage or loss of the liver is rapidly fatal.
[0056] The term "hepatic progenitor cell" as used herein refers to
a cell which can differentiate in a cell of hepatic lineage, such a
liver parenchymal cell. e.g., a hepatocyte. Hepatocytes are some of
the most versatile cells in the body. Hepatocytes have both
endocrine and exocrine functions and synthesize and accumulate
certain substance, detoxify others and secrete others to perform
enzymatic, transport, or hormonal activities. The main activities
of liver cells include bile secretion, regulation of carbohydrate
lipid, and protein metabolism, storage of substances important in
metabolism; degradation and secretion of hormones, and
transformation and excretion of drugs and toxins. The hepatic
progenitor cells of the invention can also be cultured prior to
administration to a subject under conditions which promote cell
proliferation and differentiation.
[0057] The term "hematopoietic cells" herein refers to fully
differentiated myeloid cells such as erythrocytes or red blood
cells, megakaryocytes, monocytes, granulocytes, and eosinophils, as
well as fully differentiated lymphoid cells such as B lymphocytes
and T lymphocytes. Thus, a hematopoietic stem/progenitor cell
includes the various hematopoietic precursor cells from which these
differentiated cells develop, such as BFU-E (burst-forming
units-erythroid), CFU-E (colony forming unit-erythroid), CFU-Meg
(colony forming unit-megakaryocyte), CFU-GM (colony forming
unit-granulocyte-monocyte), CFU-Eo (colony forming
unit-eosinophil), and CFU-GEMM (colony forming
unit-granulocyte-erythrocyte-megakaryocyte-monocyte).
[0058] Certain terms being set out above, it is noted that one
aspect of the present invention features a method for isolating
progenitor cells from micro-organ explants, e.g., ductal tissue
explants. A salient feature of the subject method concerns the use
of defined explants as sources from which discrete progenitor cell
populations can be amplified. For instance, as described below, the
progenitor source ductal tissue explants preferably are derived
with dimensions that allow the explanted tissue to maintain its
microarchitecture and biological function for prolonged periods of
time in culture, e.g., the dimensions of the explant preserve the
normal tissue architecture and at least a portion of the normal
tissue function that is present in vivo. Such tissue explants can
be maintained, for instance in minimal culture media for extended
periods of time (e.g., for 21 days or longer) and can be contacted
with different factors. Accordingly, carefully defined conditions
can be acquired in the culture so as selectively activate discrete
populations of cells in the tissue explant. The progenitor cells of
the present invention can be amplified, and subsequently isolated
from the explant, based on a proliferative response upon, for
example, addition of defined growth factors or biological extracts
to the culture.
[0059] In general, the method of the present invention can be used
to isolate progenitor cells from a bile duct explant by steps
beginning with the culturing of an isolated population of cells
having a microarchitecture of a mammalian bile duct, e.g. a
micro-organ explant in which the original epithelial-mesenchymal
microarchitecture of the originating duct is maintained, wherein
the dimensions of the explant provide the isolated population of
cells as maintainable in culture for at least twenty-four hours and
includes in the population of cells at least one progenitor cell
which can proliferate under such culture conditions. The explant is
contacted with an agent. e.g., a mitogenic agent such as a growth
factor or other biological extract, which agent causes
proliferation of progenitor cells in the cultured population.
Subsequently, progenitor cells from the explant that proliferate in
response to the agent are isolated, such as by direct mechanical
separation from the rest of the explant or by dissolution of all or
a portion of the explant and subsequent isolation of the progenitor
cell population.
[0060] In a illustrative embodiment, the size of the particular
ductal explant will depend largely on (i) the availability of
tissue, and (ii) a need for similar availability of nutrients to
all cells in the tissue by diffusion. In a preferred embodiment,
the ductal tissue explant is selected to provide diffusion of
adequate nutrients and O.sub.2 to every cell in a three dimensional
organ. Accordingly, the size of the explant is determined by the
requirement for a minimum level of accessibility to each cell
absent specialized delivery structures or synthetic substrates.
[0061] A salient feature of the micro-organ cultures used to in the
subject methods, according to the invention, is the ability to
preserve the cellular microenvironment found in vivo for a ductal
tissue. The invention is based, in part, upon the discovery that
under certain circumstances growth of cells of both a stromal and
epithelial layer, provided together in the same explant, will
sustain active proliferation of cells of each layer. Moreover, the
cell-cell and cell-matrix interactions provided in the explant
itself are sufficient to support cellular homeostasis. e.g.,
maturation, differentiation and segregation of cells in the explant
culture, thereby sustaining the microarchetecture and function of
the explant for prolonged periods of time.
[0062] An example of physical contact between a cell and a
noncellular substrate (matrix) is the physical contact between an
epithelial cell and its basal lamina. An example of physical
contact between a cell and another cell includes actual physical
contact maintained by, for, example, intercellular cell junctions
such as gap junctions and tight junctions. Examples of functional
contact between one cell and another cell includes electrical or
chemical communication between cells. In addition, many cells
communicate with other cells via chemical messages, e.g., hormones,
which either diffuse locally (paracrine signalling and autocrine
signalling), or are transported by the vascular system to more
remote locations (endocrine signalling).
[0063] Not wishing to be bound by any particular theory, this
microarchitecture of the ductal explants can be extremely important
for the maintenance of the explant in minimal media, e.g.; without
exogenous sources of serum or growth factors, as the ductal
explants can apparently be sustained in such minimal media by
paracrine factors resulting from specific cellular interactions
within the sample. Moreover, there is also a possibility that
certain growth factors might act indirectly by activating cells
other than progenitor cells, to produce mitogenic factors that
subsequently cause proliferation of progenitor cells within the
explant. Accordingly, the ductal explants are derived such that
they comprise both an epithelial layer and a stromal layer and
maintain in vitro the physical and/or functional interaction
between these two component of the explant.
[0064] However, the phrase "maintain, in vitro, the physical and/or
functional interaction" is not intended to exclude an isolated
population of cells in which at least one cell develops physical
and/or functional contact with at least one cell or noncellular
substance with which it is not in physical and/or functional
contact in vivo. An example of such a development is of course
proliferation of at least one cell of the isolated population of
cells.
[0065] As emphasized through the present application, the
micro-organ cultures used to prepare progenitor cells according to
the invention preserve the normal tissue architecture that is
present in vivo, e.g., the original epithelial-mesenchymal
organization. In preferred embodiments the populations of cells of
the ductal explants are grouped in a manner that preserves the
natural affinity of one cell to another, e.g., to preserve layers
of different cells present in explant. Such an association
facilitates intercellular communication. Many types of
communication takes place among animal cells. This is particularly
important in differentiating cells where induction is defined as
the interaction between one (inducing) and another (responding)
tissue or cell, as a result of which the responding cells undergo a
change in the direction of differentiation. Moreover, inductive
interactions occur in embryonic and adult cells and can act to
establish and maintain morphogenetic patterns as well as induce
differentiation (Gurdon (1992) Cell 68: 185-199). Accordingly, an
exemplary micro-organ cultures prepared in accordance to use in the
progenitor amplification method of the invention are described in
Example 1 and include a epithelial and mesenchymal cells grouped in
a manner that includes a plurality of layers so as to preserve the
natural affinity and interaction of one cell to another in and
between each layer.
[0066] In addition to isolating a ductal explant which retains the
cell-cell and cell-matrix architecture of the originating duct, the
dimensions of the explant are important to the viability of the
cells therein, e.g., where the micro-organ culture is intended to
be sustained for prolonged periods of time, e.g., 7-21 days or
longer. Accordingly, the dimensions of the explant are selected to
provide diffusion of adequate nutrients and gases (e.g., O.sub.2,
CO.sub.2, etc) to every cell in the three dimensional micro-organ
explant as well as diffusion of cellular waste out of the explant
so as to minimize cellular toxicity and concommitant death due to
localization of the waste in the micro-organ. Thus, in addition to
the requirement of both epithelial and mesenchymal components the
size of the explant is determined by the requirement for a minimum
level of accessibility to each cell in the absence specialized
delivery structures or synthetic substrates. As described herein,
this accessibility can be maintained if Aleph, an index calculated
from the thickness and the width of the explant is at least greater
than approximately 1.5 mm.sup.-1. As used herein, a surface to area
index, "Aleph" is defined as Aleph=1/x+1/a>1.5 mm.sup.-1;
wherein x=radial thickness and a=the axial length of the duct
explant in millimeters. Accordingly, the present invention provides
that the surface area to volume index of the tissue explant is
maintained within a selected range. This selected range of surface
area to volume indice provides the cells access to nutrients and to
avenues of waste disposal by diffusion in a manner similar to cells
of the in vivo organ from which the explant originated.
[0067] Examples of Aleph are provided in Table I wherein for
example, an explant having a thickness (x) of 0.1 mm and a width
(a) of 1 mm would have an Aleph index of 11. In another instance,
if x=0.3 mm and a=4 mm, the Aleph is 3.48 mm.sup.-1. To further
illustrate, Applicant has observed that when x is varied and a is
constant at 4 mm, the proliferative activity of cells in a cultured
explant is substantially reduced as the thickness of the explant
increases. Accordingly, at 900 .mu.m thickness, the number of
proliferating cells in a micro-organ culture was found to about 10
fold less than in tissue from a similar source having a thickness
of 300 .mu.m. The Aleph index for a tissue having a thickness of
900 .mu.m is 1.36 mm.sup.-1, below the minimum described herein
whereas the Aleph index for tissue having a thickness of 300 .mu.m
is 3.58 mm.sup.-1 which is well within the range defined
herein.
1TABLE I Different values for the surface area to volume ratio
index "Aleph", as a function of a (width) and x (thickness) in
mm.sup.-1 1
[0068] Again, not wishing to be bound by any particular theory, a
number of factors provided by the three-dimensional culture system
may contribute to its success in the subject method of activating
progenitor cell populations:
[0069] (a) The appropriate choice of the explant size, vis--vis the
use of the above Aleph calculations, three-dimensional matrix
provides appropriate surface area to volume ratio for adequate
diffusion of nutrients to all cells of the explant, and adequate
diffusion of cellular waste away from all cells in the explant.
[0070] (b) Because of the three-dimensionality of the matrix,
various cells continue to actively grow, in contrast to cells in
monolayer cultures, which grow to confluence, exhibit contact
inhibition, and cease to grow and divide. The elaboration of growth
and regulatory factors by replicating cells of the explant may be
partially responsible for stimulating proliferation and regulating
differentiation of cells in culture.
[0071] (c) The three-dimensional matrix retains a spatial
distribution of cellular elements, e.g., the epithelial-mesenchymal
micoarchitecture which closely approximates that found in the
counterpart tissue in vivo.
[0072] (d) The cell-cell and cell-matrix interactions may allow the
establishment of localized microenvironments conducive to cellular
induction and/or maturation. It has been recognized that maintenace
of a differentiated cellular phenotype requires not only
growth/differentiation factors but also the appropriate cellular
interactions. The present invention effectively mimics the
microenvironment. Accordingly, the micro-organ preserves
interactions which may be required to maintain the cells supporting
the progenitor cells, the cells (if any) providing inductive
signals to the progenitor cells, and the progenitor cells
themselves.
[0073] To further illustrate, the appended examples demonstrate
that microorgan explants, in which the original
epithelial-mesenchymal microarchitecture of the common bile duct
are prepared by transverse sectioning of the duct every 300 .mu.m,
can be maintained in simple media (DMEM) for several weeks and can
be used to isolate progenitor cells of the present invention by
growth induction upon contact with certain growth factors.
[0074] There are a large number of tissue culture media that exist
for culturing tissue from animals. Some of these are complex and
some are simple. While it is expected that the ductal explants may
grow in complex media, it will generally be preferred that the
explants be maintained in a simple medium, such as Dulbecco's
Minimal Essential Media (DMEM), in order to effect more precise
control over the activation of certain progenitor populations in
the explant. Furthermore, although the cultures may be grown in a
media containing sera or other biological extracts such as
pituitary extract, it has been demonstrated that neither sera nor
any other biological extract is required for explants derived
according to the above considerations (see U.S. Ser. No.
08/341,409,). Moreover, the explants can be maintained in the
absence of sera for extended periods of time. In preferred
embodiments of the invention, the growth factors or other mitogenic
agents are not included in the primary media for maintenance of the
cultures in vitro, but are used subsequently to cause proliferation
of distinct populations of progenitor cells. See the appended
examples.
[0075] The tissue explants may be maintained in any suitable
culture vessel, such as a 12 or 24 well microplate, and may be
maintained under typical culture conditions for cells isolated from
the same animal. e.g., such as 37.degree. C. in 5% CO.sub.2. The
cultures may be shaken for improved aeration, the speed of shaking
being, for example, 12 rpm.
[0076] In order to isolate progenitor cells from the ductal
explants, it will generally be desirable to contact the explant
with an agent which causes proliferation of one or more populations
of progenitor cells in the explant. For instance, a mitogen, e.g.,
a substance that induces mitosis and cell transformation can be
used to detect a progenitor cell population in the explant and
where desirable, to cause the amplification of that population. To
illustrate, a purified or semi-purifed preparation of a growth
factor can be applied to the culture. Induction of progenitor cells
which respond to the applied growth factor can be detected by
proliferation of the progenitor cells. However, as described below,
amplification of the population need not occur to a large extent in
order to use certain techniques for isolating the responsive
population.
[0077] In yet other embodiments, the ductal explants and/or
amplified progenitor cells can be cultured on feeder layers. e.g.,
layers of feeder cells which secrete inductive factors or polymeric
layers containing inductive factors. For example, a matrigel layer
can be used to induce hematopoietic progenitor cell expansion, as
described in the appended examples. Matrigel (Collaborative
Research. Inc., Bedford, Mass.) is a complex mixture of matrix and
associated materials derived as an extract of murine basement
membrane proteins, consisting predominantly of laminin, collagen
IV, heparin sulfate proteoglycan, and nidogen and entactin was
prepared from the EHS tumor as described Kleinman et al, "Basement
Membrane Complexes with Biological Activity", Biochemistry, Vol. 25
(1986), pages 312-318. Other such matrixes can be provided, such as
Humatrix. Likewise, natural and recombinantly engineered cells can
be provided as feeder layers to the instant cultures.
[0078] Methods of measuring cell proliferation are well known in
the art and most commonly include determining DNA synthesis
characteristic of cell replication. There are numerous methods in
the art for measuring DNA synthesis, any of which may: be used
according to the invention. In an embodiment of the invention. DNA
synthesis has been determined using a radioactive label
(3H-thymidine) or labeled nucleotide analogues (BrdU) for detection
by immunofluorescence.
[0079] However, in addition to measuring DNA synthesis,
morphological changes can be, and preferably will be relied on as
the basis for isolating responsive progenitor cell populations. For
instance as described in the appended examples we have observed
that certain growth factors cause amplification of progenitor cells
in ductal explants so as to form structures that can be easily
detected by the naked eye or microscopy. In an exemplary embodiment
those progenitor cells which respond to growth factors by
proliferation and subsequent formation of outgrowths from the
explant. e.g., buds or blebs can be easily detected. In another
illustrative embodiment, other structural changes, e.g., changes in
optical density of proliferating cells, can be detected via
contrast microscopy.
[0080] Various techniques may be employed to isolate the activated
progenitor cells of treated explant. Preferred isolation procedures
for progenitor cells are the ones that result in as little cell
death as possible. For example, the activated progenitor cells can
be removed from the explant sample by mechanical means, e.g.,
mechanically sheared off with a pipette. In other instances, it
will be possible to dissociate the progenitor cells from the entire
explant or sub-portion thereof, e.g., by enzymatic digestion of the
explant, followed by isolation of the activated progenitor cell
population based on specific cellular markers, e.g., using affinity
separation techniques or fluorescence activated cell sorting
(FACS).
[0081] To further illustrate, the examples below demonstrate that
ductal explants contain growth factor responsive progenitor cell
types. It is further demonstrated that different growth factors can
induce/amplify distinct populations of progenitor cells within the
ductal tissue explant to proliferate. This indicates the presence
of specific growth factor receptors on the surface of distinct
progenitor cell populations. This is important because the
expression of these receptors marks the progenitor cell populations
of interest. Monoclonal antibodies are particularly useful for
identifying markers (surface membrane proteins. e.g., receptors)
associated with particular cell lineages and/or stages of
differentiation. Procedures for separation of the subject
progenitor cell may include magnetic separation, using antibody
coated magnetic beads, affinity chromatography, and "panning" with
antibody attached to a solid matrix. e.g., plate, or other
convenient technique. Techniques providing accurate separation
include fluorescence activated cell sorting, which can have varying
degrees of sophistication, e.g., a plurality of color channels, low
angle and obtuse light scattering detecting channels, impedance
channels, etc.
[0082] Conveniently, the antibodies may be conjugated with markers,
such as magnetic beads, which allow for direct separation, biotin,
which can be removed with avidin or streptavidin bound to a
support, fluorochromes, which can be used with a fluorescence
activated cell sorter or the like, to allow for ease of separation
of the particular cell type. Any technique may be employed which is
not unduly detrimental to the viability of the cells.
[0083] In an illustrative embodiment, some of the antibodies for
growth factor receptors that exist on the subject progenitor cells
are commercially available (e.g., antibodies for EGF receptors, FGF
receptors and/or TGF receptors), and for other growth factor
receptors, antibodies can be made by methods well known to one
skilled in the art. In addition to using antibodies to isolate
progenitor cells of interest, one skilled in the art can also use
the growth factors themselves to label the cells, for example, to
permit "panning" processes.
[0084] Upon isolation, the progenitor cells of the present
invention can be further characterized in the following manner:
responsiveness to growth factors, specific gene expression,
antigenic markers on the surface of such cells and/or basic
morphology.
[0085] For example, extent of growth factor responsivity, e.g., the
concentration range of growth factor to which they will respond to,
the maximal and minimal responses, and to what other growth factors
and conditions to which they might respond, can be used to
characterize the subject progenitor cells.
[0086] Furthermore, the isolated progenitor cells can be
characterized by the expression of genes known to mark the
developing (i.e., stem or progenitor) cells for liver, pancreas,
gall bladder and intestine.
[0087] In an illustrative embodiment, the hepatocyte nuclear factor
(HNF) transcription factor family, e.g., HNF-1-4 are known to be
expressed in various cell types at various times during liver and
pancreas development. For example, the progenitor cell may express
one or more HNF protein such as HNF1.alpha., HNF1.beta.,
HNF3.beta., HNF3.gamma., and/or HNF4. ATBF1, a regulator of
alphafetoprotein (AFP) gene expression (AFP is expressed early in
liver development and is reexpressed in many liver carcinomas) is
believed to be a transient marker of liver stem cells. The LIM type
homeobox genes, such as Islet-1, are also known to be expressed
during liver development. Glut2 is a marker for both early
pancreatic and liver cells. Certain of the "forkhead" transcription
factors, such as fkh-1 or the like, are understood to be markers in
early gut tissue. Likewise, members of the CCAAT-enhancer binding
protein (C/EBP) family, such as C/EBP-.beta., are markers for early
liver development. Therefore, expression of one or more of these
genes can be used to further characterize the hepatic progenitor
cells.
[0088] In another illustrative embodiment, homeodomain type
transcription factors such as STF-1 (also known as IPF-1, IDX-1 or
PDX) have recently been shown to mark different populations of the
developing pancreas. Some LIM genes have also been shown to
regulate insulin gene expression and would also be markers for
protodifferentiated .beta. islet cells. Likewise, certain of the
PAX genes, such as PAX6, are expressed during pancreas formation
and may be used to characterize certain pancreatic progenitor cell
populations. Other markers of pancreatic progenitor cells include
the pancreas specific transcription factor PTF-1, and hXBP-1 and
the like. Moreover, certain of the HNF proteins, are expressed
during early pancrease development and may used as markers for
pancreatic progenitor cells.
[0089] In the intestine, even though there are not very many
lineage specific transcription factors that have been mapped to the
gut, the exception is the Hox B (homeobox genes) gene family (and
possibly others) which are regional type markers rather than cell
type specific but which can be used to characterize progenitor
cells originating form the specific region. e.g., the gut. Elastase
is known to be any early marker of duodenal development and hence
can be a candidate early marker for subject progenitor cells.
[0090] Certain of the subject hematopoietic progenitor cells may be
characterized by expression of such markers as c-kit, CD34 and/or
CD33, as well as OV-6, CAM5.2 and/or cytokeratin type 7 or 18.
[0091] The subject progenitor cells can also be characterized on
the basis of specific antigenic markers or other markers that may
be expressed on the cell surface, e.g., integrins, lectins,
gangliosides, or transporters, or on the basis of specific cellular
morphology. All of these techniques are known and available to the
one skilled in the art. For example, certain of the subject hepatic
and ductal progenitor cells may express the oval cell marker OV-6.
Other progenitor cells isolated from the ductal microorgan cultures
may additionally, or alternatively, express one or more markers
including hematopoietic markers, cytokeratins (such as type 7 or
19), mucin Span-1, OC.2, OC.3 or .gamma.-glutamyl transpeptidase.
Progenitor cells giving rise to pancreatic cells may express such
as markers as villin and/or tyrosine hydroxylase as well as secrete
such factors as insulin, glucagon and/or neuropeptide Y.
[0092] Once isolated and characterized, the subject progenitor
cells can be cultured under conditions which allow further
differentiation into specific cell lineages, e.g., hepatic,
pancreatic, gallbladder, intestinal, or hematopoietic lineages.
This can be achieved through a paradigm of induction that can be
developed. For example, the subject progenitor cells can be
recombined with the corresponding embryonic tissue to see if the
embryonic tissue can instruct the adult cells to codevelop and
codifferentiate. Alternatively, the progenitor cells can be
contacted with one or more growth or differentiation factors which
can induce differentiation of the cells. For instance, the cells
can be treated with a TGF.beta., such as DVR sub-family member.
[0093] Furthermore, it has become apparent, from the prolonged
viability of the explanted bile duct fragments in minimal media,
that the tissues making up the ductal explants are themselves
producing certain factors. e.g. paracrine and/or autocrine.
Accordingly, such conditioned media generated by the explants in
culture can be used to further maintain the progenitor cells in
culture subsequent to isolation from the explant. The subject
progenitor cells can be cultured in contact with the corresponding
bile duct explant, or in the conditioned media produced by such
explant.
[0094] In another preferred embodiment, the subject progenitor
cells can be implanted into one of a number of regeneration models
used in the art, e.g., partial pancreatectomy or streptozocin
treatment of a host animal.
[0095] Accordingly, another aspect of the present invention
pertains to the progeny of the subject progenitor cells, e.g. those
cells which have been derived from the cells of the initial explant
culture. Such progeny can include subsequent generations of
progenitor cells, as well as lineage committed cells. e.g.,
hepatic, pancreatic or gallbladder cells generated by inducing
differentiation of the subject progenitor cells after their
isolation from the explant, e.g., induced in vitro.
[0096] Yet another aspect of the present invention concerns
cellular compositions which include, as a cellular component,
substantially pure preparations of the subject progenitor cells, or
the progeny thereof. Cellular compositions of the present invention
include not only substantially pure populations of the progenitor
cells, but can also include cell culture components. e.g., culture
media including amino acids, metals, coenzyme factors, as well as
small populations of non-progenitor cells, e.g., some of which may
arise by subsequent differentiation of isolated progenitor cells of
the invention. Furthermore, other non-cellular components include
those which render the cellular component suitable for support
under particular circumstances, e.g., implantation, e.g.,
continuous culture.
[0097] As common methods of administering the progenitor cells of
the present invention to subjects particularly human subjects,
which are described in detail herein, include injection or
implantation of the cells into target sites in the subjects the
cells of the invention can be inserted into a delivery device which
facilitates introduction by, injection or implantation, of the
cells into the subjects. Such delivery devices include tubes, e.g.,
catheters for injecting cells and fluids into the body of a
recipient subject. In a preferred embodiment, the tubes
additionally have a needle, e.g., a syringe through which the cells
of the invention can be introduced into the subject at a desired
location. The progenitor cells of the invention can be inserted
into such a delivery device, e.g., a syringe, in different forms.
For example, the cells can be suspended in a solution or embedded
in a support matrix when contained in such a delivery device. As
used herein, the term "solution" includes a pharmaceutically
acceptable carrier or diluent in which the cells of the invention
remain viable. Pharmaceutically acceptable carriers and diluents
include saline aqueous buffer solutions solvents and/or dispersion
media. The use of such carriers and diluents is well known in the
art. The solution is preferably sterile and fluid to the extent
that easy syringability exists. Preferably, the solution is stable
under the conditions of manufacture and storage and preserved
against the contaminating action of microorganisms such as bacteria
and fungi through the use of, for example, parabens, chlorobutanol,
phenol, ascorbic acid, thimerosal, and the like. Solutions of the
invention can be prepared by incorporating progenitor cells as
described herein in a pharmaceutically acceptable carrier, or
diluent and, as required, other ingredients enumerated above
followed by filtered sterilization.
[0098] Support matrices in which the progenitor cells can be
incorporated or embedded include matrices which are
recipient-compatible and which degrade into products which are not
harmful to the recipient. Natural and/or synthetic biodegradable
matrices are examples of such matrices. Natural biodegradable
matrices include plasma clots. e.g., derived from a mammal, and
collagen matrices. Synthetic biodegradable matrices include
synthetic polymers such as polyanhydrides, polyorthoesters, and
polylactic acid. Other examples of synthetic polymers and methods
of incorporating or embedding cells into these matrices are known
in the art. See e.g., U.S. Pat. No. 4,298,002 and U.S. Pat. No.
5,308,701. These matrices provide support and protection for the
fragile progenitor cells in vivo and are, therefore, the preferred
form in which the progenitor cells are introduced into the
recipient subjects.
[0099] The present invention also provides substantially pure
progenitor cells which can be used therapeutically for treatment of
various disorders associated with insufficient functioning of the
pancreas or liver.
[0100] To illustrate, the subject progenitor cells can be used in
the treatment of a variety of pancreatic disorders both exocrine
and endocrine. For instance, the progenitor cells can be used to
produce populations of differentiated pancreatic cells for repair
subsequent to partial pancreatectomy, e.g., excision of a portion
of the pancreas. Likewise, such cell populations can be used to
regenerate or replace pancreatic tissue loss due to,
pancreatolysis, e.g., destruction of pancreatic tissue, such as
pancreatitis, e.g., a condition due to autolysis of pancreatic
tissue caused by escape of enzymes into the substance.
[0101] In an exemplary embodiment, the subject progenitor cells can
be provided for patients suffering from any insulin-deficiency
disorder. For instance, each year, over 728,000 new cases of
diabetes are diagnosed and 150.000 Americans die from the disease
and its complications; the total yearly cost in the United States
is over 20 billion dollars (Langer et al. (1993) Science 260:
920-926). Diabetes is characterized by pancreatic islet destruction
or dysfunction leading to loss of glucose control. Diabetes
mellitus is a metabolic disorder defined by the presence of
chronically elevated levels of blood glucose (hyperglycemia).
Insulin-dependent (Type 1) diabetes mellitus ("IDDM") results from
an autoimmune-mediated destruction of the pancreatic .beta.-cells
with consequent loss of insulin production, which results in
hyperglycemia. Type 1 diabetics require insulin replacement therapy
to ensure survival. Non-insulin-dependent (Type 2) diabetes
mellitus ("NIDDM") is initially characterized by hyperglycemia in
the presence of higher-than-normal levels of plasma insulin
(hyperinsulinemia). In Type 2 diabetes, tissue processes which
control carbohydrate metabolism are believed to have decreased
sensitivity to insulin. Progression of the Type 2 diabetic state is
associated with increasing concentrations of blood glucose, and
coupled with a relative decrease in the rate of glucose-induced
insulin secretion.
[0102] The primary aim of treatment in both forms of diabetes
mellitus is the same namely, the reduction of blood glucose levels
to as near normal as possible. Treatment of Type 1 diabetes
involves administration of replacement doses of insulin. In
contrast, treatment of Type 2 diabetes frequently does not require
administration of insulin. For example, initial therapy of Type 2
diabetes may be based on diet and lifestyle changes augmented by
therapy with oral hypoglycemic agents such as sulfonylurea. Insulin
therapy may be required, however, especially in the later stages of
the disease, to produce control of hyperglycemia in an attempt to
minimize complications of the disease, which may arise from islet
exhaustion.
[0103] More recently, tissue-engineering approaches to treatment
have focused on transplanting healthy pancreatic islets, usually
encapsulated in a membrane to avoid immune rejection. Three general
approaches have been tested in animal models. In the first a
tubular membrane is coiled in a housing that contained islets. The
membrane is connected to a polymer graph that in turn connects the
device to blood vessels. By manipulation of the membrane
permeability, so as to allow free diffusion of glucose and insulin
back and forth through the membrane, yet block passage of
antibodies and lymphocytes, normoglycemia was maintained in
pancreatectomized animals treated with this device (Sullivan et al.
(1991) Science 252:718).
[0104] In a second approach, hollow fibers containing islet cells
were immobilized in the polysaccharide alginate. When the device
was place intraperitoneally in diabetic animals, blood glucose
levels were lowered and good tissue compatibility was observed
(Lacey et al. (1991) Science 254:1782).
[0105] Finally, islets have been placed in microcapsules composed
of alginate or polyacrylates. In some cases, animals treated with
these microcapsules maintained normoglycemia for over two years
(Lim et al. (1980) Science 210:908; O'Shea et al. (1984) Biochim.
Biochys. Acta. 840:133; Sugamori et al. (1989) Trans. Am. Soc.
Artif. Intern. Organs 35:791; Levesque et al. (1992) Endocrinology
130:644; and Lim et al. (1992) Transplantation 53:1180). However,
all of these transplantation strategies require a large, reliable
source of donor islets.
[0106] The pancreatic progenitor cells of the invention can be used
for treatment of diabetes because they have the ability to
differentiate into cells of pancreatic lineage, e.g., .beta. islet
cells. The progenitor cells of the invention can be cultured in
vitro under conditions which can further induce these cells to
differentiate into mature pancreatic cells, or they can undergo
differentiation in vivo once introduced into a subject. Many
methods for encapsulating cells are known in the art. For example,
a source of .beta. islet cells producing insulin is encapsulated in
implantable hollow fibers. Such fibers can be pre-spun and
subsequently loaded with the .beta. islet cells (Aebischer et al.
U.S. Pat. No. 4,892,538; Aebischer et al. U.S. Pat. No. 5,106,627;
Hoffman et al. (1990) Expt. Neurobiol. 110: 39-44; Jaeger et al.
(1990) Prog. Brain Res. 82: 41-46; and Aebischer et al. (1991) J.
Biomech. Eng. 113: 178-183), or can be co-extruded with a polymer
which acts to form a polymeric coat about the .beta. islet cells
(Lim U.S. Pat. No. 4,391,909; Sefton U.S. Pat. No. 4,353,888:
Sugamori et al. (1989) Trans. Am. Artif. Intern. Organs 35:79-799:
Sefton et al, (1987) Biotehnol. Bioeng. 29:1135-1143; and Aebischer
et al. (1991) Biomaterials 12: 50-55).
[0107] Moreover, in addition to providing a source of implantable
cells, either in the form of the progenitor cell population of the
differentiated progeny thereof, the subject cells can be used to
produce cultures of pancreatic cells for production and
purification of secreted factors. For instance, cultured cells can
be provided as a source of insulin. Likewise, exocrine cultures can
be provided as a source for pancreatin.
[0108] The liver is an organ that is vulnerable to a wide variety
of metabolic, circulatory, toxic, microbial, and neoplastic
insults, and is, therefore, one of the most frequently injured
organs in the body. However, because the function of liver is very
complex, synthetically recreating its function is practically
impossible. One way of restoring liver function is by whole organ
transplantation. Although transplantation of whole liver is often
times successful it has plateaued at about 2200 transplants per
year because of donor scarcity. Therefore, alternative treatments
concentrate on manipulating the smallest functional unit of the
liver, the individual hepatocyte.
[0109] In yet another embodiment, the subject progenitor cells, or
their progeny, can be used in the treatment various hepatic
disorders. Vulnerable to a wide variety of metabolic, circulatory,
toxic, microbial, and neoplastic insults, the liver is one of the
most frequently injured organs in the body. In some instances, the
disease is primarily localized in liver cells. For example, primary
liver diseases include hereditary disorders such as Gilbert's
Syndrome, Crigler-Najjar Syndrome (either Type I or Type II). Dubin
Johnson Syndrome, familial hypercholesterolemia (FH), ornithine
transcarbamoylase (OTC) deficiency, hereditary emphysema and
hemophilia; viral infections such as hepatitis A. B. and non-A,
non-B hepatitis; and hepatic malignancies such as hepatocellular
carcinoma. Robbins, S. L. et al. (1984) Pathologic Basis of Disease
(W. B. Saunders Company, Philadelphia) pp. 884-942. More often, the
hepatic involvement is secondary, often to some of the most common
diseases of man, such as cardiac decompensation, disseminated
cancer, alcoholism, and extrahepatic infections. Robbins, S. L. et
al. (1984) Pathologic Basis of Disease (W. B. Saunders Company,
Philadelphia) pp. 884-942.
[0110] Whole liver transplantation, which is the current therapy
for a variety of liver diseases, has been employed to successfully
reconstitute LDL receptors in individuals with FH, thereby lowering
serum cholesterol to normal levels. Whole liver transplantation,
however, is limited by the scarcity of suitable donor organs. Li,
Q. et al. (1993) Human Gene Therapy 4:403-409; Kay, M. A. et al.
(1992) Proc. Natl. Acad. Sci. 89: 89-93. In addition to the
difficulty in obtaining donor organs, the expense of liver
transplantation, estimated at approximately $200.000 to $300,000
per procedure prohibits its widespread application. Another
unsolved problem is graft rejection. Foreign livers and liver cells
are poorly tolerated by the recipient and are rapidly destroyed by
the immune system in the absence of immunosuppressive drugs. Li. Q.
et al. (1993) Human Gene Therapy 4: 403-409; Bumgardner, G. L. et
al. (1992) Transplantation 53: 857-862. While immunosuppressive
drugs may be used to prevent rejection, they also block desirable
immune responses such as those against bacterial and viral
infections, thereby placing the recipient at risk of infection.
[0111] The hepatic progenitor cells of the invention can be used
for treatment of many liver disorders because they have the ability
to differentiate into cells of hepatic lineage, e.g., hepatocytes.
The progenitor cells of the invention can be cultured in vitro
under conditions which can further induce these cells to
differentiate into mature hepatocytes, or they can undergo
differentiation in vivo once introduced into a subject. Many
methods for encapsulating cells are known in the art, as has been
described above for pancreatic cells. The hepatic progenitor cells
can also be introduced directly into a subject, as they have the
potential to induce liver regeneration.
[0112] Yet another aspect of the present invention provides methods
for screening various compounds for their ability to modulate
growth, proliferation or differentiation of distinct progenitor
cell populations from bile duct tissue. A micro-organ explant that
closely mimics the properties of a given set of tissue in vivo
would have utility in screening assays in which compounds could be
tested for their ability to modulate one of growth, proliferation
or differentiation of progenitor cells in such tissue. Requirements
of a reproducible model for screening might include consistency in
the micro-architecture e.g. epithelial-mesenchymal interactions,
and nutritional environment in vitro, as well as prolonged
viability and proliferation of cultures beyond 24 hours to observe
threshold effects of compounds being screened. This level of
consistency cannot be achieved in the presence of undefined media
supplements such as sera or tissue extracts that vary between
batches and cannot be adequately controlled. The dependence of a
model on external growth supplements such as growth factors is also
undesirable as growth factors or hormones may be included among the
compounds to be tested.
[0113] In an illustrative embodiment, the ductal explants, which
maintain their microarchitecture in culture e.g., they preserve the
normal epithelial-mesenchymal architecture that is present in vivo,
can be used to screen various compounds or natural products. Such
explants can be maintained in minimal culture media for extended
periods of time (e.g., for 21 days or longer) and can be contacted
with any compound. e.g., small molecule or natural product, e.g.,
growth factor to determine the effect of such compound on one of
cellular growth, proliferation or differentiation of progenitor
cells in the explant. Detection and quantification of growth,
proliferation or differentiation of these cells in response to a
given compound provides a means for determining the compound's
efficacy at inducing one of the growth proliferation or
differentiation in a given ductal explant. Methods of measuring
cell proliferation are well known in the art and most commonly
include determining DNA synthesis characteristic of cell
replication. There are numerous methods in the art for measuring
DNA synthesis, any of which may be used according to the invention.
In an embodiment of the invention, DNA synthesis has been
determined using a radioactive label (.sup.1H-thymidine) or labeled
nucleotide analogues (BrdU) for detection by immunofluorescence.
The efficacy of the compound can be assessed by generating dose
response curves from data obtained using various concentrations of
the compound. A control assay can also be performed to provide a
baseline for comparison. Identification of the progenitor cell
population(s) amplified in response to a given test agent can be
carried out according to such phenotyping as described above.
EXEMPLIFICATION
[0114] The invention now being generally described, it will be more
readily understood by reference to the following examples which are
included merely for purposes of illustration of certain aspects and
embodiments of the present invention, and are not intended to limit
the invention.
[0115] The common bile duct (CBD) is a structure whose
developmental origins is poorly understood. Its primary function is
the delivery of bile acids from the liver to the duodenum to aid in
the emulsification of food in the digestive process, 80% of its
length traverses through the pancreas and is connected into the
pancreatic ductal system through a series of anastamoses. Through
these anastamoses and into the CBD flow the digestive enzymes
secreted by the exocrine pancreas. The relation of the CBD to the
liver and pancreas, and its morphology, are therefore essential to
both liver and pancreas normal function.
[0116] Since it contributes to both pancreatic and hepatic
function, the question arises as to whether the the CBD arises
during development as primarily a hepatic structure, a pancreatic
structure or both. Little has been done to resolve this issue in
the past due in part to a lack of early markers as well as
interest. However, there are a number of reports in the literature
citing circumstantial evidence that the adult animal retains within
the gut system stem cells for both liver and pancreas.
[0117] Because the CBD serves a dual hepato-pancreatic function, it
might be possible that it also possesses a dual identity, and might
then retain within its structure stem cells for both the pancreas
and liver. To test this hypothesis directly we isolated the CBD and
its attendant main ducts, and cultured them in vitro as intact duct
segments. The goal of this work was to study the duct as an
intact-physiological unit and to determine whether resident stem
cells could be activated to give rise to either liver and/or
pancreatic derivatives. This unique culture system allows us the
ability to study the interaction of the mesenchyme with the
epithelium, rather than the isolated culture systems of others, and
is hence more representative of the in vivo situation.
[0118] Using our unique culture system we derived cultures of
ductal fragments from the common bile duct of the adult rat in a
combination of matrigel and IGF-1 in serum free conditions induced
the formation of liver specific cell types. The liver identity of
the induced cells was determined by immunohistochemical detection
of the expression of alphafetoprotein (AFP), albumin, and ATBF-1, a
transcription factor shown to regulate AFP expression. In addition,
the formation of red blood cell clusters was observed. The
induction of albumin and AFP positive cell types was specific for
the matrigel/IGF-1 combination; culture on plastic or collagen with
IGF-1 failed to induce the appearance of these cell types.
Likewise, the substitution of IGF-1 with either TGF.alpha., EGF,
IGF-II, PDGF, or FGF.beta., all failed to elicit liver formation,
even in the presence of matrigel. However, in all of the matrigel
conditions tried, we observed the formation of red blood cell
clusters. Our results indicate that there exist both liver and
hematopoietic stem cells resident in the CBD system, that red blood
cell formation can be stimulated by a factor present in matrigel,
and that the combination of matrigel with IGF-1 can induce the
formation of liver specific cell types.
[0119] Preparation of Ductal Explants
[0120] 6 week old female Spague/Dawley from Taconic were
exsanguinated by CO.sub.2. The common bile duct and associated
pancreatic ducts was removed and placed in cold Dulbecco's Minimal
Essential Medium (DMEM) supplemented with 2 mM glutamine and
penicillin/streptomycin (100 units/ml). The duct was then cleaned
of associated pancreatic tissue, liver tissue, fat, and blood
vessels. The clean and intact duct was sectioned into approximately
250 um transverse slices, such that the original
epithelial-mesenchymal microarchitecture was retained, and placed
in medium on ice.
[0121] 250 .mu.l of reduced growth factor Matrigel (Collaborative
Research) was added to each well of a 24-well plate and incubated
at 37.degree. C. for 30 minutes. The plates were then removed and
allowed to stand at room temperature for 15 minutes. The CBD
microorgan explants were placed onto the matrigel per well and
stood at room temperature for 15 minutes to allow the ducts to
adhere to the matrigel, 1 ml of DMEM (P/S/L-glut) with or without
growth factor addition was added to the wells. Growth factors used
in the studies described below (EGF, TGF.alpha., PDGF, FGF.beta.,
FGF.alpha., IGF-1, and IGF-II) were all obtained from PreproTech.
Cultures were incubated at 37.degree. C. in a 5% CO.sub.2
atmosphere. Cultures were fed once a week with fresh medium.
[0122] Immunohistochemistry
[0123] The cultures were fixed overnight at 4.degree. C. in 4%
paraformaldehyde (PFA). The cultures were rinsed three times 10
minutes each with phosphate-buffered saline (PBS) prior to addition
of 1% periodic acid to remove endogenous peroxidase activity. The
cultures were incubated with 3% milk in PBS/0.1% Triton X-100
(PBST) for 30 minutes at room, temperature. Primary antibody to
alphafetoprotein and albumin (Accurate) were added at 1:250 for 1
hour at room temperature. ATBF-1 was used at 1:1000. The tissue was
washed 3 times with PBST over a period of 2 hours. The secondary
biotinylated antibody (Vector) was added at 1:500 for 1 hour at
room temperature. The tissue was then washed 3 times for 1 hour
with PBST and incubated with horseradish peroxidase-conjugated to
avidin (Vector) for 30 minutes at room temperature.
Antibody-binding was detected by the addition of DAB/H.sub.2O.sub.2
(Gibco). After termination of the colorimetric reaction with
H.sub.2O, 80% glycerol in PBS was added to clear the cultures prior
to photograph.
[0124] Growth Factors
[0125] Effectiveness of the growth factors in stimulating
proliferation was judged by the incorporation of Bromodeoxyuridine
(BrdU) into DNA by the responding cells. Antibodies to BrdU were
used to visualize and characterize the short term responses (24-48
hr).
[0126] The long term response was judged by the ability of these
populations of cells to be grown and expanded in cell culture as a
result of specific growth factor addition.
[0127] Different growth factors (EGF, TGF-.alpha., bFGF, aFGF,
IGF-I, IGF-II, VEGF and HGF) were used. In addition to the results
for EGF, TGF-.alpha. and bFGF described below. IGF-I. IGF-II, VEGF
and HGF were demonstrated to cause expansion of certain
subpopulations of the ductal explant.
[0128] 1. Administration of EGF to the CBD Explant
[0129] EGF was administered in three different doses 1 ng/ml, 10
ng/ml and 100 ng/ml to the CBD explant. Activation of proliferation
as assessed by BrdU labeling occurred with administration of 10
ng/ml of growth factor EGF within a span of 24 hr (FIGS. 1 and 2).
There was no difference observed between 10 and 100 ng/ml dose.
Addition of EGF to the CBD tissue explant resulted in proliferation
of distinct cells within the explant and resulted in clustering of
these cells.
[0130] 2. Administration of TGF.alpha. to the CBD Explant
[0131] TGF.alpha. was administered in the same doses as EGF.
Activation of proliferation as assessed by BrdU labeling occurred
with administration of 100 ng/ml of growth factor TGF-.alpha.
within a span of 24 hr (FIGS. 1 and 2). Unlike EGF, administration
of TGF-.alpha. to the CBD explant resulted in proliferation of
cells throughout the explant.
[0132] 3. Administration of FGF.beta. to the CBD Explant
[0133] FGF.beta. was administered in the same doses as the above
described growth factors. Activation of proliferation as assessed
by BrdU labeling occurred with administration of 10 ng/ml of
growth-factor FGF.beta. within a span of 24 hr (FIGS. 1 and 2).
Administration of 10 ng/ml of FGF.beta. resulted in induction of
distinct CBD structures to synchronously divide, implying organized
regulation of proliferative potential and response. FIG. 3 depicts
in vitro growth and expansion of CBD explant. e.g., formation of
outgrowths, e.g., blebs, in response to administration of
FGF.beta..
[0134] Preliminary long term growth experiments indicate that there
does exist a large proliferative potential within the CBD explant
that can be maintained in culture for at least 21 days.
[0135] Characterization of Expanded Progenitor Cell Populations
[0136] Antibodies that recognize transcription factors known to be
expressed during pancreas and liver formation were used to
characterize the population distribution in CBDs. STF-1, also known
as IPF-1, has been shown to be critical in pancrease formation;
Pax-6 has been shown to be critical in pancreatic endocrine cells;
Islet-1 is expressed in early gut endoderm and in islet tissue; and
HNF3.beta. is expressed in early gut endoderm and in liver
precursor cells in the endoderm. ATBF-1, Lim1/2 were not detected
in the CBD. Table 1 shows the average number of positive nuclei for
each marker per 20.times. field, or approximately every 500 .mu.m
duct length.
2TABLE 1 Scoring of Marker Distribution in the CBD cryostat
sections Markers # of nuclei Average STF-1 209 226 170 300 Pax-6 26
18 10 18 HNF3.beta. 55 50 46 50 Islet-1 33 38 42 38
[0137] HNF3.beta. is an early marker for endoderm formation and is
one of the earliest markers expressed by the developing liver.
Sections of CBD were stained for the early endoderm and liver
marker HNF3.beta.. The common bile duct (CBD) contains cells that
are immunopositive for HNF3.beta.. Its expression in the CBD is
sporadic but specific to the epithelium, as expected. HNF3.beta. is
expressed throughout the CBD and also in the main pancreatic ducts,
although less frequently. Positive cells were often found in
clusters of epithelial cells that were branching away from the main
lumen of the CBD. HNF3.beta.-expressing cells were found uniformly
throughout the CBD. Because of HNF3.beta.'s role as an early
endoderm and liver marker in embryonic development, we hypothesized
that these. HNF3.beta. expressing cells might be liver stem cells
resident in the adult hepatopancreatic duct system.
[0138] If the observed HNF3.beta. expressing cells are indeed liver
precursors one might then use ductal cultures to attempt to in
vitro activate the formation of liver-specific cell types and
structures. Construction of such a culture system would then allow
the study of the signals and interactions required to induce the
liver development program. To this end we cultured duct fragments
that had been transversely cut into approximate 250 .mu.m
widths.
[0139] These were then cultured either on plastic, collagen,
directly on feeder layers (STO, C3H10T1/2), or on matrigel
(Collaborative Research, Inc. Bedford, Mass.). Matrigel was the
only matrix permutation tried that gave liver formation.
Furthermore, the formation of liver structures only occurred with
the addition of IGF-1. Addition of FGF.beta., TGF.alpha., EGF,
IGF-II, PDGF-AA all failed to induce the formation of liver
structures.
[0140] The addition of IGF-1 to duct cultures gave rise to three
basic duct colonies; "Nonresponders" in which there was little
observed growth or change in morphology (FIG. 4A), "N-type"
colonies which underwent dramatic changes in morphology to give
rise to neuritelike processes which were in fact fibroblasts laying
end on end (FIG. 4B) and "L-type" colonies which took on a
liver-like morphology, with epithelial blebs and the formation of
red blood cell clusters (FIG. 4C). This liver-like morphology
resembles that of cultured embryonic liver (data not shown). The
number and frequency of the formation of all three colony types is
documented in the table of FIG. 5.
[0141] Because of the morphological similarity between our L-type
colonies and cultured embryonic liver, we decided to look for the
expression of liver-specific markers in all three colony types. The
markers used for this analysis were alphafetoprotein (AFP),
albumin, and ATBF-1. All three have been shown to be specific for
early liver. In all cases examined the only cultures which stained
immunopositive for all three markers were the L-type colonies.
[0142] For example, cultures were stained for the expression of the
early liver marker AFP. AFP is expressed within 24 hours of liver
formation in embryos, and is lost in adult hepatic tissue. Our data
indicated extensive expression of AFP in IGF-1 treated duct
fragments. We also saw loose cells that were highly positive for
AFP. These tended to be large cells in diameter. We also observed
the formation of duct-like structures that have arisen in the CBD
explants. These duct-like structures were often surrounded by
highly positive AFP staining cells. Some of the duct-like
structures appear themselves to be AFP positive, probably due to
the fact that AFP is a secreted factor.
[0143] Cultures were also stained for the early liver marker
ATBF-1. ATBF-1 has been shown to regulate expression of AFP during
embryonic liver development. We observed staining of L-type
colonies with the liver marker ATBF-1.
[0144] All of the above-cited references and publications are
hereby incorporated by reference.
Equivalents
[0145] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
following claims.
* * * * *