U.S. patent application number 12/289019 was filed with the patent office on 2009-02-26 for processes for clonal growth of hepatic progenitor cells.
This patent application is currently assigned to University of North Carolina at Chapel Hill. Invention is credited to Hiroshi Kubota, Lola M. Reid.
Application Number | 20090053758 12/289019 |
Document ID | / |
Family ID | 46280550 |
Filed Date | 2009-02-26 |
United States Patent
Application |
20090053758 |
Kind Code |
A1 |
Kubota; Hiroshi ; et
al. |
February 26, 2009 |
Processes for clonal growth of hepatic progenitor cells
Abstract
A method of propagating mammalian endodermally derived
progenitors such as hepatic progenitors, their progeny, or mixtures
thereof is developed which includes culturing mammalian
progenitors, their progeny, or mixtures thereof on a layer of
embryonic mammalian feeder cells in a culture medium. The culture
medium can be supplemented with one or more hormones and other
growth agents. These hormones and other growth agents can include
insulin, dexamethasone, transferrin, nicotinamide, serum albumin,
.beta.-mercaptoethanol, free fatty acid, glutamine, CuSO.sub.4, and
H.sub.2SeO.sub.3. The culture medium can also include antibiotics.
Importantly, the culture medium does not include serum. The
invention includes means of inducing the differentiation of the
progenitors to their adult fates such as the differentiation of
hepatic progenitor cells to hepatocytes or biliary cells by adding,
or excluding epidermal growth factor, respectively. The method of
producing mammalian progenitors is useful in that the progenitors
can be used subsequently in one or more of the following processes:
identification of growth and differentiation factors, toxicological
studies, drug development, antimicrobial studies, or the
preparation of an extracorporeal organ such as a bioartificial
liver.
Inventors: |
Kubota; Hiroshi; (Chapel
Hill, NC) ; Reid; Lola M.; (Chapel Hill, NC) |
Correspondence
Address: |
FOLEY AND LARDNER LLP;SUITE 500
3000 K STREET NW
WASHINGTON
DC
20007
US
|
Assignee: |
University of North Carolina at
Chapel Hill
|
Family ID: |
46280550 |
Appl. No.: |
12/289019 |
Filed: |
October 17, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10135700 |
May 1, 2002 |
7456017 |
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12289019 |
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09679663 |
Oct 3, 2000 |
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10135700 |
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60157003 |
Oct 1, 1999 |
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Current U.S.
Class: |
435/32 ; 435/29;
435/350; 435/353; 435/354; 435/363; 435/370 |
Current CPC
Class: |
C12N 2502/13 20130101;
C12N 2500/25 20130101; C12N 2501/39 20130101; C12N 5/0672 20130101;
C12N 2500/90 20130101; C12N 2500/20 20130101; C12N 2500/36
20130101; C12N 2501/11 20130101 |
Class at
Publication: |
435/32 ; 435/29;
435/370; 435/363; 435/350; 435/353; 435/354 |
International
Class: |
C12Q 1/18 20060101
C12Q001/18; C12Q 1/02 20060101 C12Q001/02; C12N 5/06 20060101
C12N005/06 |
Claims
1. A method of identifying hepatic growth factors comprising
providing hepatic progenitors, their progeny, or mixtures thereof,
and a sample suspected of comprising at least one hepatic growth
factor, combining the progenitors, their progeny, or mixtures
thereof, and the sample, and observing stimulation of growth of the
hepatic progenitors.
2. A method of identifying hepatic differentiation factors
comprising providing hepatic progenitors, their progeny, or
mixtures thereof, and a sample suspected of comprising at least one
hepatic differentiation factor, combining the progenitors, their
progeny, or mixtures thereof, and the sample, and observing
differentiation of the hepatic progenitors.
3. A method of identifying a hepatic toxin comprising providing
hepatic progenitors, their progeny, or mixtures thereof, and a
sample suspected of comprising at least one hepatic toxin,
combining the progenitors, their progeny, or mixtures thereof, and
the sample, and observing the death of the hepatic progenitors.
4. A method of developing a drug comprising providing hepatic
progenitors, their progeny, or mixtures thereof, and a drug
suspected of a capacity to affect hepatic progenitor, metabolisms
and combining the progenitors, their progeny, or mixtures thereof,
and the sample, and observing a change in the metabolism of the
hepatic progenitors.
5. A method of identifying novel antimicrobials comprising
providing hepatic progenitors, their progeny, or mixtures thereof,
and at least one agent suspected of having an antimicrobial effect,
combining the progenitors, their progeny, or mixtures thereof, and
the agent, and observing changes in growth of the hepatic
progenitors.
6. A method of preparing an extracorporeal liver comprising
providing hepatic progenitors, their progeny, or mixtures thereof
and culturing the progenitors, their progeny, or mixtures thereof
in a bioreactor until a sufficient population is obtained effective
to serve as an extracorporeal liver.
7. A method of propagating hepatic progenitors, their progeny, or
mixtures thereof comprising: (a) providing at least one hepatic
progenitor and (b) culturing the hepatic progenitor in a culture
medium comprising at least one feeder cell biosynthetic
product.
8. The method of claim 7 in which the culture medium further
comprises glucocorticoid, insulin, epidermal growth factor,
nicotinamide, or combinations.
9. The method of claim 7 in which the feeder cell biosynthetic
product comprises a peptide, a glycopeptide, a lipopeptide, a
lipid, a glycolipid, a carbohydrate, a protein, a glycoprotein, a
lipoprotein, or combinations thereof, in which the biosynthetic
product enhances the proliferation or differentiation of the
hepatic progenitor.
10. The method of claim 7 in which the feeder cell biosynthetic
product comprises insulin-like growth factors, interleukin-6 family
growth factors, hepatocyte growth factor, fibroblast growth factor,
extracellular matrix, or combinations thereof, in which the
biosynthetic product enhances the proliferation or differentiation
of the hepatic progenitor.
11. The method of claim 7 in which the at least one hepatic
progenitor comprises at least one cell from human, non-human
primate, pig, dog, rabbit, rat, or mouse.
12. The method of claim 7 which further comprises cloning the at
least one hepatic progenitor.
13. The method of claim 7 further comprising adding a growth
factor.
14. The method of claim 13 further comprising adding a
differentiation factor to produce hepatocytes, biliary cells, or
combinations thereof.
Description
[0001] This application is a divisional of U.S. patent application
Ser. No. 10/135,700, filed May 1, 2002, which is a continuation of
U.S. patent application Ser. No. 09/679,663, filed Oct. 3, 2000,
which claims priority from U.S. Provisional Application No.
60/157,003, filed Oct. 1, 1999, all of which are incorporated
herein by reference.
1. FIELD OF THE INVENTION
[0002] The present invention relates to novel conditions for clonal
growth of mammalian hepatic progenitors, including pluripotent
cells, stem cells, and other early hepatic progenitor cells. In
particular, the invention relates to methods of propagating hepatic
progenitor cells using defined culture medium and feeder cells in
co-cultures. Moreover, the invention relates to the cells used as
feeders and capable of sustaining hepatic progenitor cell
growth.
2. DESCRIPTION OF RELATED ART
[0003] Identification of multipotential progenitor cell populations
in mammalian tissues is important both for clinical and commercial
interests and also for understandings of developmental processes
and tissue homeostasis. Progenitor cell populations are ideal
targets for gene therapy, cell transplantation and for tissue
engineering of bioartificial organs (Millar, AD. 1992 Nature 357,
455; Langer, R. and Vacanti, J. P. 1993 Science 260, 920; Gage, F.
H. 1998 Nature 392, 18).
[0004] The existence of tissue-specific, "determined" stem cells or
progenitors having high growth potential and/or pluripotentiality
is readily apparent from studies on hematopoietic stem cells
(Spangrude, G. J. et al. 1988 Science 241, 58), neuronal stem cells
(Davis, A. A., and Temple, S. 1994 Nature 372, 263; Stemple, D. L.,
and Anderson, D. J. 1992 Cell 71, 973) and epidermal stem cells
(Jones, P. H. and Watt, F. M. 1993 Cell 73, 713), each having been
identified clonally by using the particular methods appropriate for
that tissue. These progenitors are regarded as the cells
responsible for normal hematopoietic, neuronal or epidermal tissue
homeostasis and for regenerative responses after severe injury
(Hall, P. A., and Watt, F. M. 1989 Development 106, 619).
[0005] The mammalian adult liver has a tremendous capacity to
recover after either extensive hepatotoxic injury or partial
hepatectomy (Fishback, F. C. 1929 Arch. Pathol. 7, 955); (Higgins,
G. M. and Anderson, R. M. 193 Arch. Pathol. 12, 186), even though
the liver is usually a quiescent tissue without rapid turnover.
Data from recent studies in the mouse have been interpreted to
suggest that adult parenchymal cells have an almost unlimited
growth potentiality as assayed by serial transplantation
experiments (Overturf et al. 1997 Am. J. Pathol. 151, 1273); (Rhim,
J. A. et al. 1994 Science 263, 1149). These experiments made use of
heterogeneous liver cell populations limiting the ability to prove
that the growth potential observed derived from adult parenchymal
cells, from a subpopulation of adult parenchymal cells and/or from
immature stages of the parenchymal cells (i.e. progenitors).
Furthermore, the studies show no evidence for biliary epithelial
differentiation, since the hosts used had either albumin-urokinase
transgenes or, in the other case, a tyrosine catabolic enzyme
deficiency; both types of hosts have conditions that selected for
the hepatocytic lineage. Therefore, the assay was incapable of
testing for bipotent cell populations.
[0006] Several histological studies establish that early hepatic
cells from midgestational fetuses have a developmental
bipotentiality to differentiate to bile duct epithelium as well as
to mature hepatocytes (Shiojiri, N. 1997 Microscopy Res. Tech. 39,
328-35). Hepatic development begins in the ventral foregut endoderm
immediately after the endodermal epithelium interacts with the
cardiogenic mesoderm (Douarin, N. M. 1975 Medical Biol. 53, 427);
(Houssaint, E. 1980 Cell Differ. 9, 269). This hepatic commitment
occurs at embryonic day (E) 8 in the mouse. The initial phase of
hepatic development becomes evident with the induction of serum
albumin and alpha-fetoprotein mRNAs in the endoderm and prior to
morphological changes (Gualdi, R. et al. 1996 Genes Dev. 10, 1670).
At E 9.5 of mouse gestation, the specified cells then proliferate
and penetrate into the mesenchyme of the septum transversum with a
cord-like fashion, forming the liver anlage. Although the liver
mass then increases dramatically, the increase in mass is due
largely to hematopoietic cells, which colonize the fetal liver at
E10 in the mouse (Houssaint, E. 1981 Cell Differ. 10, 243) and
influence the hepatic cells to show an extremely distorted and
irregular shape (Luzzatto, A. C. 1981 Cell Tissue Res. 215, 133).
Interestingly, recent data from gene-targeting mutant mice
indicates that impairment of a number of genes has led to lethal
hepatic failure, apoptosis and/or necrosis of parenchymal cells
between E12 to E15 (Gunes, C. et al. 1998 EMBO J. 17, 2846);
(Hilberg, F. et al. 1993 Nature 365, 1791); (Motoyama, J. et al.
1997 Mech. Dev. 66, 27); (Schmidt, C. et al. 1995 Nature 373, 699).
Especially gene disruptions that are part of the stress-activated
cascade (Ganiatsas, S. et al. 1998 Proc. Natl. Acad. Sci. USA 95,
6881); (Nishina, H. et al. 1999 Development 126, 505) or
anti-apoptotic cascade (Beg, A. et al. 1995 Nature 376, 167); (Li,
Q. et al. 1999 Science 284, 321); (Tanaka, M. et al. 1999. Immunity
10, 421) can result in severely impaired hepatogenesis, not
hematopoiesis, in spite of the broad expression of the inactivated
gene. It isn't clear whether hepatic cells are intrinsically
sensitive to developmental stress stimuli or that the particular
microenvironment in fetal liver per se causes such destructive
effects (Doi, T. S. et al 1999 Proc. Natl. Acad. Sci. USA 96,
2994). On the other hand, the basic architecture of adult liver is
dependent on the appearance of the initial cylinder of bile duct
epithelium surrounding the portal vein (Shiojiri, N. 1997
Microscopy Res. Tech. 39, 328). Immunohistologically, the first
sign of the differentiation of intrahepatic bile duct epithelial
cells is the expression of biliary-specific cytokeratin (CK). CK
proteins, the cytoplasmic intermediate filament (IF) proteins of
epithelial cells, are encoded by a multigene family and expressed
in a tissue- and differentiation-specific manner (Moll, R. et al.
1982 Cell 31, 11). CK19 is one of the most remarkable biliary
markers, because adult hepatocytes don't express CK19 at all,
whereas adult biliary epithelial cells do express this protein.
Only CK8 and CK18 are expressed through early hepatic cells to
adult hepatocytes (Moll, R. et al. Cell 1982, 31, 11. At E15.5 in
the rat development, corresponding to E14 in the mouse, the biliary
precursors are heavily stained by both CK18 and CK8 antibodies, and
some biliary precursors express CK19. As development progresses,
maturing bile ducts gradually express CK7 in addition to CK19 and
lose the expression of ALB (Shiojiri, N. et al. Cancer Res. 1991,
51, 2611). Although hepatic cells as early as E13 in the rat are
thought to be a homogeneous population, it remains to be seen
whether all early hepatic cells can differentiate to biliary
epithelial cell lineage, and how their fates are determined.
Definitive lineage-marking studies, such as those using retroviral
vectors, have not been done for hepatic cells, and clonal culture
conditions requisite for the demonstration of any bipotent hepatic
progenitor cells have not been identified.
[0007] For clonal growth analyses, one major obstacle is the
explosive expansion of hematopoietic cells, marring the ability to
observe ex vivo expansion of hepatic cells. Therefore an enrichment
process for the hepatic population must be used. Although the
surface markers needed to fractionate the hematopoietic cells in
fetal liver have been investigated in detail (Dzierzak, E. et al.
Immunol. Today 1998, 19, 228), those for hepatic progenitor cells
are still poorly defined, since the studies are in their infancy
(Sigal, S. et al. Hepatology 1994, 19, 999). Furthermore, the ex
vivo proliferation conditions typically used for adult liver cells
result in their dedifferentiation with loss of tissue-specific
functions such as ALB expression (Block, G. D. et al. J. Cell Biol.
1996, 132, 1133). A somewhat improved ability to synthesize
tissue-specific mRNAs and a restoration in the ability to regulate
tissue-specific genes fully post-transcriptionally occurs only in
liver cells maintained in the absence of serum and with a defined
mixture of hormones, growth factors and/or with certain
extracellular matrix components (Jefferson, D. M. et al. Mol. Cell.
Biol. 1984, 4, 1929; Enat, R. et al Proc. Natl. Acad. Sci., 1984,
81, 1411). Proliferating fetal hepatic cells, however, maintain the
expression of such serum proteins in vivo. What has not been clear
in the field is how to maintain and grow hepatic progenitors in
vitro. There is an unfilled need for identification of conditions
that sustain the ex vivo expansion of hepatic progenitor cells.
Likewise there is an unfilled need for an in vitro colony forming
assay (CFA) for defining clonal growth potential of hepatic
progenitors freshly isolated from liver tissue; clonal growth is
defined as the ability of a single cell seeded into culture being
able to generate a population of daughter cells that are clonally
derived from the seeded cell. Others have described colony growth
(Block, G. D. et al. J. Cell Biol. 1996, 132, 1133), consisting of
aggregates of cells growing closely together in liver cultures
seeded at high cell densities; however, the colonies of cells
described in these prior studies could not be subcultured and,
therefore, by definition were not clonal and of limited
utility.
[0008] Others have attempted to grow hepatocytes in vitro. U.S.
Pat. No. 5,510,254 to Naughton et al. claims the culture of
hepatocytes depends on a three-dimensional framework of
biocompatible but non-living material. There is an unfilled need
for hepatocyte culture conditions where no artificial framework is
necessary and that provides the condition for hepatic progenitors
to be expanded and cultured. Furthermore, there is a need for
cloned hepatic progenitors with bipotential differentiation
capability, that is ability to generate both biliary and
hepatocytic lineages, and suitability for use as components of a
bioartificial liver, for testing of hepatotoxins and drug
development, among other uses.
[0009] U.S. Pat. No. 5,559,022 to Naughton et al., claims liver
reserve cells that bind Eosin Y, a stain that was used to
characterize the "reserve cells", but did not use well-established
markers for liver cells, nor provided methods for clonal expansion,
nor provided markers by which to isolate viable liver reserve
cells. There is an unfilled need for methods that teach how to
isolate and culture cells that have many features essential to
hepatic progenitors, including expression of at least one specific
marker and the potential to differentiate into either hepatocytes
or biliary cells. There is also an unfilled need for methods for
clonal growth of hepatic progenitors. Clonal growth is essential as
a clear and rigorous distinction and identification of pluripotent
hepatic progenitors.
[0010] U.S. Pat. No. 5,405,772 to Ponting claims a culture medium
for cell growth. The U.S. Pat. No. 5,405,772 requires the use of
3-30 .mu.g/ml cholesterol, 5-30 .mu.g/ml nucleosides, and either
2-100 .mu.g/cm2 collagen IV or 0.5-100 .mu.g/cm2 fibronectin. There
is a need for a culture medium that is specific for, and optimized
for, hepatic progenitor cell growth.
[0011] U.S. Pat. No. 4,914,032 to Kuri-Harcuch et al. claims a
process for culturing hepatocytes. In contrast to the instant
invention, U.S. Pat. No. 4,914,032 fails to teach either the
culture of hepatic progenitors or clonal growth conditions for
hepatic cells. Likewise, U.S. Pat. No. 5,030,105 to Kuri-Harcuch et
al. claims methods of assessing agents by treating hepatocyte
cultures. There is an unfilled need for clonal growth conditions so
that defined populations of cells may be used for testing and also
for methods for the culture of hepatic progenitors.
[0012] The U.S. Pat. No. 5,858,721 to Naughton et al. claims
transfection of stromal cells. The U.S. Pat. No. 5,858,721 patent
is limited, however, by the requirement for a framework of
biocompatible, non-living material. The instant invention by
contrast, there is an unfilled need for growth conditions that do
not require a synthetic meshwork.
[0013] The present inventors have recognized the inadequacy of
growing mature liver cells, such as hepatocytes, rather than the
far more useful hepatic progenitors. They have carefully defined
the isolation parameters for hepatic progenitors and requirements
for clonal growth. The progenitor cells and the methods for
selecting and culturing the progenitors have many uses, including
utility in medicine for treatment of patients with liver failure,
and utility for evaluation of toxicity agents, and utility for
evaluation of drugs.
[0014] U.S. Pat. Nos. 5,576,207 and 5,789,246 to Reid, et al. teach
the need for feeders and a hormone-supplemented defined medium.
These prior studies advocated use of embryonic liver stromal cells
in combination with defined extracellular matrix substrata, and a
serum-free, hormonally defined medium as conditions for expansion
of hepatic progenitors. However, the defined medium used was more
complex than the one used by the instant invention; the cells were
plated onto purified matrix substrata (type IV collagen and
laminin), whereas here they are plated directly onto the feeders
(that supply that matrix); and the embryonic stromal cells were
prepared as primary cultures of embryonic livers and were not
established as cell lines. By use of embryonic stromal cell lines,
the feeder cells are provided by a far easier, more practical and
more reproducible means of supporting the cells. Moreover, it is
reasonable to assume that the STO feeders will not restrict support
to just hepatic progenitors but can be used for progenitors from
multiple tissue types. The prior patent, the hepatic progenitor
cultures were seeded at high cell densities and expansion of them
was observed as colony formation, meaning that the aggregates of
the cells, not clones of cells, were induced to proliferate.
3. SUMMARY OF THE INVENTION
[0015] The present invention relates to a method of propagating
progenitors, their progeny, or mixtures thereof. In particular, the
present invention relates to a method of propagating
endodermally-derived progenitors, their progeny, or mixtures
thereof. The cells are derived from endodermal tissue. Then the
endodermally-derived progenitors, their progeny, or mixtures
thereof, are cultured on a layer comprising feeder cells in a
culture medium. The progenitors, their progeny, or mixtures
thereof, can be vertebrate cells. The progenitors, their progeny,
or mixtures thereof, can express the phenotype ICAM or ICAM-1
positive and classical MHC class I antigen negative. The classical
MCH class I antigen is also termed MHC class Ia antigen.
[0016] The present invention also relates to a method of culturing
hepatic stem and other progenitor cells using a serum-free,
hormone-supplemented, defined medium and feeder cells. Also, the
invention relates to a method of culturing the progeny of
progenitor cells, or combinations of progenitor cells and
progenitor progeny. Preferably, the progenitor cells are hepatic
progenitors. Likewise, the present invention relates to a method of
cloning hepatic pluripotent progenitor cells using specific culture
conditions. Preferably, the invention relates to a method of
cloning hepatic pluripotent progenitor cells. The hepatic
pluripotent progenitor cells may be derived from any invertebrate
or vertebrate species and more preferably mammalian. Even more
preferably, the hepatic pluripotent progenitor cells are human,
primate, pig, dog, rat, rabbit or mouse in origin. Most preferably
the pluripotent progenitor cells are human in origin. The invention
teaches particular culture conditions that are required for the ex
vivo expansion of hepatic progenitor cells, and their progeny. The
invention also teaches use of embryonic feeder cells, such as STO
mouse embryonic cells, as feeder cells for hepatic progenitors. The
feeder cells are used in combination with a novel serum-free,
hormonally defined medium (HDM) taught in the invention. The
combination enabled the inventors to establish various rat fetal
hepatic cell lines from E15 rat livers without malignant
transformation of the cells.
[0017] Furthermore, the invention relates to methods of cloning
feeder cells capable of sustaining propagation of hepatic
progenitor cells, and their progeny.
[0018] The invention also relates to specific cell lines that, when
used as feeders, support hepatic progenitor cell growth.
[0019] The invention additionally relates to methods of cloning
hepatic progenitor cells. The invention teaches the use of the
hepatic cell lines and the HDM-STO co-culture system for
development of an in vitro colony forming assay (CFA) for defining
clonal growth potential of freshly isolated hepatic progenitors.
The CFA, when combined with cells purified by specific antigenic
profile, reveals bipotent hepatic progenitors. For example,
progenitors from E13 rat livers, corresponding to E11.5 in the
mouse, and with high growth potential have the same phenotype as
classical MHC class I (RT1A1)-, OX18 (pan-MHC class I) dull, and
intracellular adhesion molecule 1 (ICAM-1)+.
[0020] The invention additionally relates to the culture medium
capable of sustaining clonal hepatic cell growth. The culture
medium features several specific hormones and nutrients and an
absence of serum.
[0021] Further still, the invention relates to the culture of
hepatic progenitors in medium with feeder cell biosynthetic
products.
[0022] The invention further relates to methods of inducing hepatic
cell differentiation, including production of hepatocyte and
biliary cell phenotypes. Epidermal growth factor (EGF) is taught in
this invention to influence both growth of the progenitor colonies
and their fates as either hepatocytes or biliary epithelial
cells.
4. BRIEF DESCRIPTION OF THE DRAWINGS
[0023] FIGS. 1A-1C are characterizations of hepatic cell lines from
day 15 fetal rat liver.
[0024] FIGS. 2A-2F are assays of colony formation on fibroblast
feeder cells.
[0025] FIGS. 3A-3X are expressions of rat cell surface antigens on
various hepatic cell lines in adult liver cells.
[0026] FIGS. 4A1-4A2, 4B1-4B5, 4C1-4C5, and 4D1-4D4 depict
phenotypic analysis of day 13 fetal rat livers.
[0027] FIGS. 5A-5D depict characterization of hepatic colonies in
the absence and presence of EGF.
[0028] FIGS. 6A-6B depict induction of CK19 expression on
RT1A1-hepatic cells.
[0029] FIG. 7 is a schematic representation of hepatic colony
formation on STO5 feeder cells.
5. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0030] The instant invention is a process for propagation and use
of stem cells. Various tissues are appropriate sources of
progenitors, including tissues of ectoderm, mesoderm and endoderm
origin. The ectoderm tissues can include skin tissue, brain tissue
and other nerve tissue. The mesoderm tissues can include muscle,
the blood and hemopoietic systems. The endoderm tissues can include
the gut, stomach, pancreas thyroid and glands associated with the
digestive system. In particular, the instant invention is a process
for the propagation of hepatic stem cells and of other hepatic
progenitor cells. The process involves exposing populations of
isolated hepatic stem cells and/or hepatic progenitor cells and/or
their progeny, to growth conditions capable of sustaining clonal
growth, that is, growth at very low cell densities. In a preferred
embodiment, the process involves using a serum-free,
hormone-supplemented, defined medium to support the propagation of
hepatic progenitor cells on a layer of feeder cells. The function
of the feeder cells is multi-fold, including supplying nutrients,
supplying an attachment surface, and secreting into the medium
certain growth factors and extracellular matrix components needed
for survival, growth and/or differentiation of the hepatic
progenitor cells. In another preferred embodiment, the process
involves selecting for cells that are capable of sustaining the
growth of hepatic stem and hepatic progenitor cells. The feeder
cells may be from reptiles, birds, crustaceans, fish, annelids,
mollusks, nematodes, insects, or mammals, preferably human.
Preferably, the feeder cells derive from embryonic tissues. Also,
preferably, the feeder cells derive from embryonic tissue. Also,
preferably, the feeder cells can derive from embryonic liver
tissue. Additionally, the feeder cells may be genetically modified.
In a still more preferred embodiment, the process involves cloning
feeder cells that optimally sustain hepatic cells.
[0031] Any method of isolating hepatic stem and hepatic progenitor
cells is acceptable, including by affinity-based interactions, e.g.
affinity panning, immunosurgery in combination with complement, by
flow cytometry, by centrifugal elutriation, by differential
centrifugation, etc. The isolated hepatic stem and progenitor
cells, have the capacity to express some or all of the phenotype
markers (classical MHC class I-, ICAM-1+, OX18dull,
alpha-fetoprotein+, or albumin+). It is another embodiment of the
invention that the hepatic progenitors express a growth pattern in
the colonies characterized by formation of piled-up cells as
aggregates, colonies or clusters.
[0032] It is a preferred embodiment of the instant invention that
hepatic cells be selectively grown in a serum-free,
hormone-supplemented, defined medium (HDM).
[0033] The composition of HDM comprises a nutrient medium
including, but not limited to a mixture of Dulbecco's modified
Eagle's medium and Ham's F12 to which is added up to about 40 ng/ml
EGF, up to about 5-10 .mu.g/ml insulin, up to about 10-6 M
Dexamethasone or other glucocorticoid hormone, up to about 10
.mu.g/ml iron-saturated transferrin, up to about 5.times.10-2M
nicotinamide, up to about 2% bovine serum albumin, up to about
5.times.10-4M 2-mercaptoethanol or equivalent reducing agent, up to
about 8 .mu.eq/l free fatty acid, up to about 2.times.10-2M
glutamine, up to about 1.times.10-6 M CuSO4, up to about
3.times.10-8 M H2SeO3 and, optionally, antibiotics. Antibiotics can
include penicillin, streptomycin, gentamycin, and others common in
the art, and combinations thereof. One skilled in the art will know
that other nutrient media, e.g. Ham's F-10, Medium 199, or one of
the MCDB series including MCDB 151 and MCDB 302, can, after minimal
testing, be used in place of DMEM/F12. The most minimal conditions
for cell expansion are use of the feeders in the absence of any
hormones; and the most critical of the hormonal requirements listed
above are glucocorticoids, insulin, transferrin, and EGF
constituting the strict hormonal mitogens for progenitor cell
expansion. Other hormonal factors can be added and might have
secondary growth effects but do not replace the critical
requirements noted above. Likewise, changes in the hormone
constituents such as can be made by one of ordinary skill in the
art, are within the scope of the instant invention.
[0034] Preferable ranges include 10-50 ng/ml EGF, 2-10 ug/ml
insulin, 5.times.10-7M to 5.times.10-6M dexamethasone
(9a-fluoro-16a-methyl-prednisolone), 5-20 ug/ml iron-saturated
transferrin, 2-8.times.10-3M nicotinamide, 0.05-0.5% serum albumin,
2-8.times.10-5 M 2-mercaptoethanol, 5-10 ueq free fatty acid
mixture, 1-3.times.10-3 M glutamine, 0.5-2.times.10-6 M CuSO4,
1-5.times.10-8M H2SeO3, 1-5 uM palmitic acid, 0.1-0.4 uM
palmitoleic acid, 0.5-1.2 uM stearic acid, 0.5-2 uM oleic acid, 1-5
uM linoleic acid, and 0.2-0.8 uM linolenic acid.
[0035] The serum-free, hormonally defined culture medium of the
invention, is suitable for the clonal growth of hepatic cells. This
HDM contains a basal medium that can be any of a number of options
such as Dulbecco's modified Eagle's medium (DME), Ham's F12, RPMI
1640, Williams E medium, etc. A preferred embodiment is a 1:1
mixture of Dulbecco's modified Eagle's medium and Ham's F12
(DMEM/F12, from, for example GIBCO/BRL, Grand Island, N.Y.). The
basal medium is supplemented with epidermal growth factor, EGF
(from, for example, Collaborative Biomedical Products) at a
preferred concentration of 10 ng/ml, insulin (from, for example,
Sigma) at a preferred concentration of 5 .mu.g/ml, 10-6M
Dexamethasone (from, for example, Sigma), 10 .mu.g/ml
iron-saturated transferrin (Sigma), 4.4.times.10-3M nicotinamide
(from, for example, Sigma), 0.2% serum albumin (from, for example,
Sigma), 5.times.10-5M 2-mercaptoethanol (from, for example, Sigma),
7.6 .mu.eq/l free fatty acid mixture (2.4 uM palmitic acid, 0.21 uM
palmitoleic acid, 0.88 uM stearic acid, 1 uM oleic acid, 2.7 uM
linoleic acid, and 0.43 uM linolenic acid), 2.times.10-3M glutamine
(from, for example, GIBCO/BRL), 1.times.10-6M CuSO4, 3.times.10-8M
H2SeO3 and antibiotics. The growth factors secreted by the feeder
cells, including but not limited to insulin-like growth factors
(IGFs), interleukin (IL)-6 family, hepatocyte growth factors
(HGFs), and fibroblast growth factors (FGFs), can be added to the
culture medium to augment feeder effects but have not been found to
replace feeder effects when added singly or in various
combinations, meaning that the feeder cells are producing other
signals, yet unidentified that are needed alone or in combination
with these growth factors.
[0036] It is a still further embodiment of the invention that the
hepatic progenitor cells are propagated from a single progenitor
cell, that is, that the cells are cloned. Growing cells in colonies
does not necessarily equate with clonal growth which implicitly and
explicitly is defined as propagation of cells derived from a single
cell. Any of several methods of cloning known in the art are
suitable, including diluting the progenitor cells to one cell, or
less, per cell culture plate well, a method termed limiting
dilution. Similarly progenitor cells may be cloned with the use of
cloning rings, by selective ablation, by dilute culture on
microparticles, by single-cell sorting using flow cytometry, by
picking individual cells with micropipet or optical tweezers, and
by agar.
[0037] It is a yet further embodiment of the invention that many of
the cloned progenitor cells are capable of mitosis. It is preferred
that the progenitor cells are capable of a least one cycle of
mitosis and even more preferred that the progenitor cells are
capable of at least ten cycles of division.
[0038] It is a still yet further embodiment of the invention that
hepatic progenitor cells and their progeny are propagated in medium
supplemented with metabolic and biosynthetic products of feeder
cells. The supplement can take the form of conditioned medium, that
is, medium previously incubated with living feeder cells.
Preferably the supplementing can take the form of isolating from
feeder cell-conditioned medium those factors including proteins,
peptides, lipids, carbohydrates, and metabolic regulators that
sustain and enhance the growth of hepatic progenitors and their
progeny. The proteins can include soluble and insoluble components
of extracellular matrix and growth factors including epidermal
growth factor and insulin-like growth factors.
[0039] It is further preferred that hepatic cells be selectively
grown in culture using a layer of feeder cells, where those feeder
cells are embryonic or adult cells or other suitable cells. In one
embodiment the feeder cells are stromal cells or fibroblasts. The
fibroblasts or other suitable cells may be genetically modified,
e.g. by transfection. It is preferred that the fibroblasts or other
suitable cells be human, non-human primate, pig, dog, rabbit, rat,
or mouse mesodermal cells, and other mammalian and avian mesodermal
cells are also suitable. Furthermore, the fibroblasts can be cloned
and selected for the ability to support hepatic progenitor
cells.
[0040] It is a preferred embodiment of the instant invention that
isolated hepatic progenitor cells be committed to a hepatocyte or
biliary cell lineage by the selective application, or absence, of
epidermal growth factor (EGF), or other differentiation signal.
[0041] It is a still more preferred embodiment of the instant
invention that isolated stem cells and other hepatic progenitor
cells be used as a component of a bioartificial liver that can be
used as an extracorporeal liver assist device. It is a still more
preferred embodiment of the instant invention that the
bioartificial liver containing isolated hepatic progenitor cells
and their progeny be used to support the life of a patient
suffering from liver malfunction or failure.
6. EXAMPLES
[0042] The following examples are illustrative of the invention,
but the invention is by no means limited to these specific
examples. The person of ordinary skill in the art will find in
these examples the means to implement the instant invention. The
person of ordinary skill in the art will recognize a multitude of
alternate embodiments that fall within the scope of the present
invention.
6.1. Preparation and Analysis of Hepatic Stem and Hepatic
Progenitor Cells
[0043] Rats. Pregnant Fisher 344 rats are obtained from Charles
River Breeding Laboratory (Wilmington, Mass.). For timed
pregnancies, animals are put together in the afternoon, and the
morning on which the plug is observed is designated day 0. Male
Fisher 344 rats (200-250 g) are used for adult liver cells.
[0044] Establishment of hepatic cell lines. Fetal livers are
prepared from day 15 of the gestation. Single cell suspensions are
obtained by incubating the livers with 0.05% trypsin and 0.5 mM
EDTA or 10 units/ml thermolysin (Sigma, St. Louis, Mo.) and 100
units/ml deoxyribonuclease I (Sigma) for at 37EC. The cells are
overlayed on Ficoll-paque (Pharmacia Biotech, Uppsala, Sweden) for
gradient density centrifugation at 450 g for 15 min. The cells from
the bottom fraction are inoculated into tissue culture dishes
coated with 17 mg/ml collagen type IV (Collaborative Biomedical
Products, Bedford, Mass.) or 12 .mu.g/ml laminin (Collaborative
Biomedical Products) for th1120-3 and rter6 or rhel4321,
respectively. The serum-free hormonally defined culture medium,
HDM, is a 1:1 mixture of Dulbecco's modified Eagle's medium and
Ham's F12 (DMEM/F12, GIBCO/BRL, Grand Island, N.Y.), to which is
added 20 ng/ml EGF (Collaborative Biomedical Products), 5 .mu.g/ml
insulin (Sigma), 10-7M Dexamethasone (Sigma), 10 .mu.g/ml
iron-saturated transferrin (Sigma), 4.4.times.10-3M nicotinamide
(Sigma), 0.2% Bovine Serum Albumin (Sigma), 5.times.10-5M
2-mercaptoethanol (Sigma), 7.6 .mu.eq/l free fatty acid,
2.times.10-3M glutamine (GIBCO/BRL), 1.times.10-6M CuSO4,
3.times.10-8M H2SeO3 and antibiotics. Each concentration given is
the final concentration in the medium. After 4 weeks of culture,
trypsinized cells are cultured on a feeder layer of mitomycin
C-treated STO mouse embryonic fibroblast line (American Type
Culture Collection, Rockville Md.). Th1120-3, rter6, and rhel4321
are cloned from three independent preparations of fetal hepatic
cells and are maintained on STO feeder cells with HDM. After the
establishment of the cell lines, the concentration of EGF is
reduced to 10 ng/ml for all cell cultures.
[0045] Cell adhesion assay. Adhesion of cells to fibronectin
(Collaborative Biomedical Products), laminin and collagen type IV
is evaluated using 96 well micro-titer plates (Corning, Cambridge,
Mass.) coated with these proteins at 0.3 to 10 .mu.g/ml. After
removing the STO cells by Percoll (Pharmacia Biotech) gradient
density centrifugation at 200 g for 15 min, 3.times.104 cells of
the hepatic cell lines, th1120-3, rter6, and rhel4321, are cultured
in each well for 10 hours with HDM. After rinsing twice to remove
floating cells, fresh medium with the tetrazolium salt WST-1
(Boehringer Mannheim, Indianapolis, Ind.) is added to measure the
number of variable adherent cells. After 4 hours, the absorbance is
determined according to the manufacturer's protocol.
[0046] STO Sublines. One hundred cells of parent STO from ATCC are
cultured in 100 mm culture dishes for 7 days in DMEM/F12
supplemented with 10% heat-inactivated fetal bovine serum,
2.times.10-3M glutamine, 5.times.10-5M 2-mercaptoethanol and
antibiotics. Four subclones are selected for further
characterization according to the cell morphology and the growth
speed. Although CFA for rter6 is performed in the four subclones,
one of them, STO6, does not persist in attaching to culture plates
after mitomycin C-treatment. One subclone, STO5, is transfected
with pEF-Hlx-MC1neo or pEF-MC1neo kindly provided from Dr. J. M.
Adams, The Walter and Eliza Hall Institute of Medical Research.
Linearized plasmids at Nde I site are introduced into cells by
DOSPER liposomal transfection reagent (Boehringer Mannheim). After
G418 selection, six clones are isolated. Three clones of each are
analyzed by CFA.
[0047] Immunohistochemical Staining of Colonies. Culture plates are
fixed in methanol-acetone (1:1) for 2 min at room temperature,
rinsed and blocked by Hanks Balanced Salt Solution (HBSS) with 20%
goat serum (GIBCO/BRL) at 4EC. For double immunohistochemistry of
alpha-fetoprotein and albumin, plates are incubated with anti-rat
albumin antibody (ICN Biomedicals, Costa Mesa, Calif.) followed by
Texas Red-conjugated anti-rabbit IgG (Vector laboratories,
Burlingame, Calif.) and FITC-conjugated anti rat alpha-fetoprotein
polyclonal antibody (Nordic Immunology, Tilburg, Netherlands). For
double labeling of albumin and CK19, anti-CK19 monoclonal antibody
(Amersham, Buckinghamshire, England) and FITC-conjugated anti mouse
IgG (Caltag, Burlingame, Calif.) are used instead of anti
alpha-fetoprotein antibody.
[0048] Dissociation of E13 of fetal liver. Fetal livers are
dissected into ice-cold Ca++ free HBSS with 10 mM HEPES, 0.8 mM
MgSO4 and 1 mM EGTA (pH7.4). The livers are triturated with 0.2%
type IV collagenase (Sigma) and 16.5 units/ml thermolysin (Sigma)
in HBSS prepared with 10 mM HEPES, 0.8 mM MgSO4, and 1 mM CaCl2.
After incubation at 37EC for 10 min, the cell suspension is
digested with 0.025% trypsin and 2.5 mM EDTA (Sigma) for 10 min.
Trypsin is then quenched by addition of 1 mg/ml trypsin inhibitor
(Sigma). Finally, the cells are treated with 200 units/ml
deoxyribonuclease I (Sigma). In all experiments, 3-5.times.105
cells per liver are obtained.
[0049] Isolation of adult liver cells. The two step liver perfusion
method is performed to isolate liver cells. After perfusion, the
cells are centrifuged for 1 min at 50 g twice to enrich for large
parenchymal cells. Cellular viability is >90% as measured by
trypan blue exclusion.
[0050] Flow cytometric analysis. Cells are analyzed on a FACScan
(Becton-Dickinson, Mountain View, Calif.) and sorted using a Moflow
Flow Cytometer (Cytomation, Fort Collins, Colo.). The cell
suspensions from E13 fetal liver are incubated with HBSS,
containing 20% goat serum (GIBCO/BRL) and 1% teleostean gelatin
(Sigma), on ice to prevent nonspecific antibody binding. After
rinsing, the cells are resuspended with FITC-conjugated anti rat
RT1Aa,b,1 antibody B5 (Pharmingen, San Diego, Calif.) and
PE-conjugated anti-rat ICAM-1 antibody 1A29 (Pharmingen). In some
experiments the cells are stained with biotinylated anti-rat
monomorphic MHC class I antibody OX18 (Pharmingen) followed by a
second staining with streptavidin-red670 (GIBCO/BRL) for 3 color
staining. All stainings are performed with ice-cold Ca++free HBSS
containing 10 mM HEPES, 0.8 mM MgSO4, 0.2 mM EGTA, and 0.2% BSA
(pH7.4). The established three hepatic cell lines are trypsinized
and fractionated by Percoll density gradient centrifugation to
remove feeder cells. The rat hepatoma cell line, FTO-2B, and the
rat liver epithelial cell line, WB-F344, as well as adult liver
cells are stained to compare with the fetal hepatic cell lines. The
cell lines are kind gifts of Dr. R. E. K. Fournier, Fred
[0051] Hutchinson Cancer Research Center, Seattle, Wash., and Dr.
M.-S. Tsao, University of North Carolina, Chapel Hill, N.C.,
respectively. Cells are blocked and stained with FITC-conjugated
B5, OX18, PE-conjugated 1A29 or anti FITC-conjugated rat integrin
.quadrature.1 antibody Ha2/5 (Pharmingen). FITC-conjugated anti
mouse IgG is used for OX18. Cell suspensions of three fetal hepatic
cell lines are stained with biotinylated anti-mouse CD98 followed
by a second staining with streptavidin-red670 as well as anti-rat
moAb to gate out mouse cell populations.
[0052] Various antigens are expressed in different relative numbers
by cells. In practical usage the level of expression of a
particular antigen can be NO expression, a low level of expression,
a level of expression that is normal or regular for many antigens,
and a high level of expression. In this usage, the term "low is
used interchangeably with a weak or dull. More detailed description
of the level of expression can, alternatively, be made, but these
four levels suffice for many purposes. It should be clear that
measurement of antigen expression by, for example, flow cytometry,
provides a continuous range for antigen expression.
[0053] CFA for hepatic cell lines, sorted cells, and adult liver
cells. The hepatic cell lines are plated in triplicate at 500 cells
per 9.6 cm2 on mitomycin C-treated STO feeder layer with the same
HDM as used for maintaining each cell line. Before plating, cell
are trypsinized and fractionated by Percoll density gradient
centrifugation to remove feeder cells. The cultures are incubated
for 10 to 14 days with medium changes every other day. Double
immunofluorescence staining of alpha-fetoprotein and albumin is
then performed. 100 colonies per well are analyzed by the colony
morphology, P or F type, and the expression of alpha-fetoprotein
and albumin. The colonies are stained using Diff-Quick (Baxter,
McGaw Park, Ill.) to count the number of the colonies per well. In
the CFA for primary sorted cells and adult liver cells, the plating
cell number is changed as described. As another minor modification,
the culture period is expanded to between 14 and 17 days, and the
concentration of dexamethasone is increased to 10-6M. All other
procedures are performed as above. In the CFA for adult liver
cells, small numbers of clumps of liver cells are not eliminated
from the cell suspension after the preparation. Therefore, an
undefined number of the colonies might be produced from the clumps.
For CFA of biliary differentiation on sorted cells, double
immunofluorescence staining of albumin and CK19 of the colonies is
performed at 5 days each of the culture in the presence or absence
of EGF. At day 5 of the cultures, any colony with more than one
CK19+ cell is counted as a CK19+ colony. At day 10 and 15, colonies
containing multiple clusters of two CK19+ cells or one cluster of
more than three CK19+ cells are counted as a CK19+ colony. About
100 colonies per well are counted. Each point represents the
mean.+-.SD from triplicate-stained cultures.
6.2. Generation and Characterization of Fetal Rat Hepatic Cell
Lines Using Feeders of Mouse Embryonic Cells with a Hormonally
Defined Medium
[0054] Simple long-term cultures of rat E15 hepatic cells are
attempted to see how long fetal hepatic cells could be maintained
and expanded ex vivo to produce progeny. After a gradient density
centrifugation to remove hematopoietic mononuclear cells, the fetal
liver cells are cultured on culture dishes coated by collagen type
IV or laminin and in HDM (see example 6.1). The cells survive well
for more than 4 weeks. However, secondary cultures on fresh
collagen type IV- or laminin-coated dishes do not permit further
expansion. When mitomycin C-treated STO embryonic mouse fibroblast
cell lines are used as a feeder layer for the secondary cultures,
many aggregates of cells grow. Eventually several stable hepatic
cell lines are established from four independent experiments.
[0055] Immunohistochemical analysis of alpha-fetoprotein and
albumin are performed in the continuous growing cell populations
before cloning of the cell lines. Both proteins, alpha-fetoprotein
and albumin, are used as the markers to confirm that cell
populations originated from the hepatic lineage. The cell
population with a tendency to form piles of cells, is called
P-colonies, and has intense expression of alpha-fetoprotein and
albumin, while the flattened monolayers, called F-colonies, have
diminished expression of alpha-fetoprotein and no albumin. The
embryonic mouse fibroblasts, STO, do not show any reactivity to
either antibody. For further analysis, three cloned hepatic cell
lines from independent experiments are selected by the
morphological criteria of either P-type colonies or F-type
colonies. Rhel4321 (FIG. 1a) consists mostly of packed small cells,
P-type colonies, whereas th1120-3 (FIG. 1c) makes only a flattened
monolayer of F-type colonies. Rter6 (FIG. 1b) is an intermediate
phenotype of these two. Interestingly, the heterogeneity of rter6
is still observed after three rounds of sequential cloning of the
flattened colony. To see the heterogeneity of colonies derived from
single cells in rhel3421 and rter6, the cells are cultured on STO
fibroblasts for 10 to 14 days at a seeding density of 500 cells per
9.6 cm.sup.2 (one well of a 6-well plate). The colonies are then
characterized in terms of their morphology and their expression of
alpha-fetoprotein and albumin. FIG. 2a to f shows the results. In
the cell lines, rhel4321 (FIG. 2b) and rter6 (FIG. 2c), and in the
original cell population prior to cloning (FIG. 2a), almost all
P-type colonies strongly express alpha-fetoprotein, whereas F-type
colonies of cells do not. Furthermore, the intense expression of
both alpha-fetoprotein and albumin is observed only in P-type
colonies. The morphological difference in the cloned hepatic cell
lines correlate to the percentage of the P-type colony (FIGS. 2b
and c). The percentage of P-type colonies in CFA of rter6 and
rhel4321 is 33.3% (8.6% SD) and 65.7% (+4.0% SD), respectively. The
total colony number per well is counted to calculate the clonal
growth efficiency (colony efficiency). The efficiency of rter6 and
rhel4321 is 45.7% (.+-.1.3% SD) and 36.4% (.+-.1.1% SD),
respectively. The th1120-3 cells tightly attach to each other along
their lateral borders making preparation of single cell suspensions
difficult. However, the th1120-3 cells do not produce piled up
clusters.
[0056] Next, the preferences of each of the cell lines to adhere to
specific components of extracellular matrices (ECM) are tested,
because the adhesion of mouse liver cells to such ECM proteins as
laminin, collagen type IV, and fibronectin, changes in different
developmental stages. Whereas collagen type IV is the most
effective in the attachment of th1120-3 (FIG. 1c), similar to the
findings for the adult liver cells, it works less well for rter6
(FIG. 1b) and rhel4321 (FIG. 1a). Laminin is the most effective
substratum for adhesion of rhel4321 (FIG. 1a). This preference is
similar to that of primary cultures of mouse fetal liver cells. In
summary, the conserved expression of alpha-fetoprotein and albumin
in P type colonies and preferential adherence to laminin by
rhel4321, suggest that the cell populations producing P type
colonies are more strictly associated with hepatic progenitor
cells.
6.3. Isolation of STO Subclones for the Colony Formation; Assay of
Hepatic Progenitors
[0057] To develop a CFA system to identify bipotent hepatic
progenitors with high growth potential, the culture system has to
be able to support cell expansion at clonal seeding densities and
with conservation of critical original hepatic functions. albumin
and alpha-fetoprotein are two of the most significant markers for
early hepatic development. The culture conditions optimizing P type
colonies should be the best, since P type, but not F type, colonies
maintain the expression of alpha-fetoprotein and albumin during
clonal expansion. Therefore, STO subclones are compared in their
support of P type colonies of rter6. One of the clones, STO5,
supports the P type colony formation more than any of the other
sublines and more than the parent line (FIG. 2d). The CFA of
rhel4321 also confirms that STO5 is a more effective feeder than
the parent STO (FIG. 2e). The mouse Hlx gene product, expressed in
the mesenchymal cells lining digestive tract from E10.5, is
essential for fetal hepatic cell expansion. Although the mRNA
expression for the Hlx gene is analyzed in all the STO subclones,
there is no significant difference in its expression among the
subclones (data not shown). Furthermore, the stable transfectants
of mouse Hlx in STO5 do not result in an improvement in the colony
formation assays (FIG. 2f). One clone of the transfectants,
however, is used for further experiments, because the transfectant
supports a more stable persistence of the original morphology of
STO5 at relatively high passages.
6.4. Identification of Hepatic Progenitors from E13 Fetal Liver
Using the Surface Antigenic Markers and the Colony Forming
assay.
[0058] Hepatopoiesis and massive amounts of hematopoiesis co-exist
in the fetal liver. So far, the antigenic profile of hematopoietic
progenitors has extensively been analyzed, whereas studies of early
hepatic progenitors are still in their infancy. The antigenic
profile of hepatic cells is analyzed using the three hepatic cell
lines established in this study, an adult hepatocarcinoma cell line
(FTO-2B), an epithelial cell line from adult rat liver (WB-F344),
and freshly isolated adult liver cells. Compared with FTO-2B,
WB-F344, and adult liver cells, the pattern of the most immature of
the fetal hepatic cell lines, rhel4321, is quite unique in that
there is no expression of classical MHC class I (RT1A.sup.1) (FIGS.
3a-3x). The cell line, th1120-3 (FIGS. 3i-3l), is similar to
rhel4321 (FIGS. 3a-3d) in the pattern of RT1A.sup.1, OX18 (pan-MHC
class I), and ICAM-1, whereas rter6 (FIGS. 3e-3h) has relatively
high expression of RT1A.sup.1 and OX18 (FIG. 3). Additionally,
another cell line from a different experiment, which has an
identical morphology to rhel4321, is also RT1A.sup.1-,
OX18.sup.dull, and ICAM-1.sup.+. Integrin b.sub.1 expression is
similar in all the cell lines, while the pattern of RT1A.sup.a,b,1
and ICAM-1 is unique among them. The antigenic profile of adult
liver cells is RT1A.sup.1+, OX18.sup.+, and ICAM-1.sup.+. Since, in
the adult rat, all bone marrow cells except mature erythrocytes
strongly express MHC class I molecules, the fetal hepatic
population can be separated from the hemopoietic cell populations
by MHC class I expression. The cell suspensions from rat E13 livers
are stained with anti RT1A.sup.1 and ICAM-1 antibodies. FIG. 4a
shows the 2 color-staining pattern of RT1A.sup.1 and ICAM-1. To
determine which fraction contains the hepatic cell population, five
fractions are isolated by fluorescent activated cell sorting and
then screened by CFA for clonal growth potential. FIG. 4b
represents the result of resorting of the five fractions after
sorting. The hepatic cell colonies, defined by expression of
albumin and alpha-fetoprotein, are distinguishable also
morphologically, enabling one to count the number of hepatic
colonies per well. The majority of the hepatic colonies are
detected in the gate RT1A.sup.1dull and ICAM-I.sup.+ (Table 1, FIG.
4b gate 2), and the frequency of the P type colony is 75.6%.+-.4.9%
SD). Gate 1 shows a much lower number of the colonies, and the
other fractions contain negligible numbers of cells with colony
forming ability. In gates 1 and 2, the expression of both
alpha-fetoprotein and albumin is confirmed in all the hepatic
colonies. Some of the colonies, derived from cells in gate 2, are
larger than others. To investigate the MHC class I expression on
the hepatic cells in detail, three color staining of RT1 A.sup.1,
ICAM-1, and OX18 with the sidescatter (SSC) as another parameter is
used for the cell fractionation. Sidescatter (SSC), a reflection of
the granularity of cell, is a useful parameter for separation of
hepatic from hematopoietic cells, because fetal hepatic cells
contain lipid droplets as early as E11 of gestation (Luzzatto,
1981). FIG. 4c shows that the gate 2 contains the highest number of
colony-forming cells. Gating R2 based on the SSC, the population
corresponding to the gate 2 clearly shows RT1A.sup.1- and
OX18.sup.dull phenotype (FIGS. 4c, 4d). The CFA confirms that R4
harbors more colony-forming cells than gate 2 (Table 1). These
results suggest that most of the RT1A.sup.1-,OX18.sup.dull, and
ICAM-1.sup.+ population from E13 rat liver are hepatic cells
producing alpha-fetoprotein.sup.+ and albumin.sup.+ colonies. It is
the identical antigenic profile found for rhel4321 cells (FIG.
3).
TABLE-US-00001 TABLE 1 The Frequency of hepatic colonies from
sorted E13 fetal liver based on the expression of RT1A and ICAM-1.
Inoculated cell (per Hepatic colony (per Efficiency of colony Gate
well) well) formation (%) 1 1000 8.7 .+-. 4.0 0.87 2 500 136.3 .+-.
4.6 27 3 5000 10.0 .+-. 7.9 0.13 4 5000 6.3 .+-. 0.6 0.13 5 5000
5.0 .+-. 1.0 0.10 R3 1000 7.0 .+-. 2.6 0.70 R4 500 269.3 .+-. 9.8
54
[0059] Colony forming culture on STO5hlx containing indicated cell
number from each fraction of E13 of fetal liver. Number of the
hepatic colonies was established from triplicate stained cultures
(mean.+-.SD). Efficiency of the colony formation express the
percentage of cells inoculated to culture that went on to form
colonies analyzed after 16 days of the culture.
6.5. Different Growth Requirement of E13 Hepatic Cells and Adult
Liver Cells
[0060] The growth requirement of the sorted hepatic cells from E13
liver are studied using the defined STO5 feeders and the HDM. EGF
has long been known as a potent growth factor for adult liver
cells. Therefore, the effects of EGF for colony formation of sorted
hepatic cells are investigated. The colony-size of the RT1A.sup.1-
OX18.sup.dull ICAM-1.sup.+ hepatic cells becomes bigger in the
absence of EGF, whereas adult liver cells yielded colonies only in
the presence of EGF (FIG. 6[a and] c). Furthermore, the morphology
of the colonies derived from adult liver cells is the typical F
type, whereas all RT1A.sup.1- hepatic cells produce P type colonies
without EGF. However, the colony efficiency is reduced slightly by
the absence of EGF (FIG. 6a). Interestingly, the culture condition
in the absence of EGF emphasized the two types of P-colonies, P1
and P2. Although the majority of the colonies is P2 type, at the
12th day of culture, it is difficult to distinguish the two types
definitively because some of them do not have the typical
morphology like FIG. 6a. These results suggest that fetal hepatic
cells and adult liver cells are intrinsically different in their
growth requirement as well as in their expression of RT1A.sup.1
(FIGS. 3 and 4) and colony morphology.
[0061] After 3 weeks of culture, when growth seems to reach a
maximum, the expression of RT1A.sup.1-, OX18, and ICAM-1 is
assessed. As shown in FIGS. 5b-5d, the expression of RT1A.sup.1 is
not induced, while that of OX18 is reduced. The level of ICAM-1
does not change. Furthermore, the average cell number of single
colony is calculated from the recovered cell number, the percentage
of rat hepatic cells and the colony efficiency. The estimated cell
number reaches 3 to 4.times.10.sup.3 (Table 2). This indicates that
the single cell forming the colonies divided approximately 11-12
times on average under this culture condition.
TABLE-US-00002 TABLE 2 Calculation of the cell number in single
hepatic colony. Average of Seeding Culture Percentage Colony cell
number Inoculated denisty length Recovered of rat cell efficiency
in single cell number (cell/cm.sup.2) (day) cell number (%) (%)
colony 500 18 18 1.5 .times. 10.sup.6 58 41 4.2 .times. 10.sup.3
4000 51 21 6.0 .times. 10.sup.6 90 44 3.1 .times. 10.sup.3 4000 51
20 4.0 .times. 10.sup.6 69 21 3.3 .times. 10.sup.3
[0062] Sorted cells from R4 in FIG. 4c were cultured on STO5hlx
feeder in 60 mm or 100 mm dish. After the period indicated of the
culture cell all cells were recovered and the total cell number
counted. The percentage of rat cells is from flow cytometric
analysis based on the expression of rat ICAM-1 and mouse CD98.
Colony efficiency indicates the percentage of cells inoculated to
culture that went on to form colonies. Data from triplicate-stained
cultures (mean) was obtained from the experiments run parallel
with.
Average of cell number in single colony=(Recovered cell
number.times.Percentage of rat cell/100)/Inoculated cell
number.times.Colony efficiency/100)
6.6. Evidence for Bipotentiality in RT1A.sup.1- Hepatic
Progenitor
[0063] At E13 of gestation in the rat, the hepatic cells are
thought to have a bipotent precursor giving rise to the mature
hepatocyte and bile duct epithelium. However, before the
discoveries of the instant invention there has been no direct
evidence whether the two fates originated from a single cell or
not. To determine whether the RT1A1-OX18dull ICAM-1+ fetal hepatic
cells can differentiate to the biliary lineage in this culture
system, the colonies are stained by anti-CK19 as a specific marker
for biliary epithelial cells. CK19 is expressed in the bile duct
epithelial precursors after day 15.5 in the fetal rat liver at
which time the expression of albumin disappears in the cells. The
sorted RT1A.sup.1- ICAM-1.sup.+ cells are cultured in the presence
or absence of EGF, and their fates are monitored by the expression
of CK19 and albumin after 5 days of culture. After the first 5
days, the CK19.sup.+ colonies are negligible in the cultures
treated with EGF, whereas a few colonies containing CK19.sup.+
cells occurred in those in the absence of EGF. Although the
intensity of the CK19 expression is fairly weak, the CK19.sup.+
cells show reduced albumin expression. At the 10th day of the
culture, some colonies apparently express only CK19 or albumin and
others have dual positive expression. The pattern of the CK19.sup.+
and albumin.sup.+ cells in a single colony is reciprocal. The
number of dual positive colonies and CK19 single positive colonies
still is higher in the absence of EGF (FIG. 6a). In the presence of
EGF, many of the colonies consist only of albumin.sup.+ cells at
the 10th day (FIG. 7b). Eventually, the percentage of dual positive
colonies reaches nearly 100% in the absence of EGF at day 15 (FIG.
6a). Altogether, EGF dramatically suppresses the appearance of
CK19.sup.+ colonies through the culture (FIG. 6b). These results
suggest that the RT1A.sup.1-, OX18.sup.dull, and ICAM-1.sup.+ cells
from E13 fetal liver can differentiate towards the biliary lineage
and their fate can be influenced by EGF in vitro (FIG. 7).
6.7. Protocol for Isolation and Cloning of Feeder Cells Capable of
Sustaining Clonal Growth of Hepatic Stem and Hepatic Progenitor
Cells.
[0064] Fresh embryonic tissue or frozen tissue (e.g. liver, lung,
kidney, muscle, intestine) from pig, beagle, rabbit, mouse or
monkey is minced in calcium-free, phosphate-buffered saline (PBS).
After rinsing with PBS a couple of times, the cell suspension is
incubated with 0.25% trypsin for 10 min at 370 or for 60 min at
room temperature with agitating using a magnetic stirrer. The
remaining cell chunks are removed by filtering the suspension
thorough mesh. The cells are then cultured on tissue culture dishes
with a basal medium (e.g. Eagle's MEM) supplemented with serum
(e.g. 10% fetal calf serum) and with any of various growth
supplements (e.g. 2 mM glutamine, sodium pyruvate, and MEM
nonessential amino acids). Plastic substratum and serum
supplemented medium are generic conditions that permit expansion of
a cell population that is a candidate as support cells ("feeder
cells"), most commonly being mesodermally-derived (e.g. stromal
cells), and that provide factors supporting the survival, growth
and/or functions of another cell type (e.g. progenitor cells). The
feeder cells are subcultured with 0.05% trypsin when they become
confluent or almost confluent. After several rounds of subculture,
expanded cells are prepared as frozen stocks and stored as such
until use. An alternative source of feeder cells can be
commercially available primary cultures of feeder cells or feeder
cell lines. In any case, the following criteria are needed to
identify the appropriate feeder cells:
[0065] The feeder cells support
[0066] 1) clonal growth of hepatic progenitors with the phenotypic
markers classical MHC class I antigen(s) negative, ICAM-1 positive,
and/or nonclassical MHC class I antigen(s) dull positive;
[0067] 2) clonal growth of progenitors with progeny with the
phenotype markers classical MHC class I antigen(s) negative, ICAM-1
positive, nonclassical MHC class I antigen(s) dull positive,
alpha-fetoprotein positive, albumin positive or CK19 positive;
or
[0068] 3) inducible differentiation into both hepatic lineage and
biliary lineage, required to define bipotent hepatic
progenitors.
[0069] In the field, classical MHC class I antigen is also known as
MHC class Ia antigen. Non-classical MHC class I antigen is also
known as MHC class Ib antigen. The MHC antigens have different
designations in different species: RT1 in rat, H-2 in mouse, and
HLA in humans, for example.
[0070] The assays noted above are described below:
[0071] A clonal growth condition for hepatic progenitors
[0072] The hepatic progenitors are plated at 500 cells per 9.6 cm2
on growth-arrested, i.e. cells treated to prevent proliferation,
feeder cells. The feeder cells are growth-arrested by treating them
with mitomycin C or by irradiating (3000-5000 rads depending upon
cell type). The growth-arrested feeder cells and progenitor cells
are fed with a serum-free HDM. As an example, HDM for the rodent
cells is a 1:1 mixture of Dulbecco's modified Eagle's medium and
Ham's F12 with added 10 ng/ml EGF, 5 .mu.g/ml insulin, 10-6M
Dexamethasone, 10 .mu.g/ml iron-saturated transferrin,
4.4.times.10-3M nicotinamide, 0.2% bovine serum albumin,
5.times.10-5M 2-mercaptoethanol, 7.6 .mu.eq/l free fatty acid,
2.times.10 3M glutamine, 1.times.10-6M CuSO4, 3.times.10-8M H2SeO3
and antibiotics. The cultures are incubated for 10 to 14 days with
medium changes every other day. Double immunofluorescence staining
of alpha-fetoprotein, albumin, and/or CK19 is then performed for
identifying the fate of the progeny. About 100 colonies are
analyzed by the expression of alpha-fetoprotein and albumin.
Furthermore the colony morphology, P or F type, could be useful
identification of the relevant progeny.
[0073] The ideal combination of feeder cells and hepatic
progenitors are those that originated from the identical species.
Preferably, the feeder cells are from the same tissue and same
species as the hepatic progenitors. However, mixing of feeders from
one species and progenitors from another is possible. For example,
even rodent feeder cells can be used for human hepatic progenitors.
Soluble and insoluble factors (that can be species- and/or
tissue-specific) help the clonal growth of hepatic stem cells or
hepatic progenitors. The source of the factors is:
[0074] Conditioned medium from the cultured feeder cells of the
optimal species and tissue. The feeder cells can be of any cell
type, not just stromal cells.
[0075] When the critical factor(s) are known, one makes a
biologically active feeder cell population by introduction into any
cells of complementary DNA or mRNA for transcription or
translation, respectively, for the synthesis of relevant molecules
(signals) derived from optimal feeder cells active for hepatic
progenitors.
[0076] If the critical factor(s) are known, one can also replace
the feeder cells altogether by supplementing the medium with those
signals, whether they be proteins, peptides, carbohydrates, lipids,
glycopeptides, glycoproteins, lipoproteins, glycolipids, or a
combination of these constituting the signals derived from optimal
feeder cells active for hepatic progenitors.
[0077] The above examples have been depicted solely for the purpose
of exemplification and are not intended to restrict the scope or
embodiments of the invention. Other embodiments not specifically
described should be apparent to those of ordinary skill in the art.
Such other embodiments are considered to fall, nevertheless, within
the scope and spirit of the present invention. Thus, the invention
is properly limited solely by the claims that follow.
[0078] All patents and publications cited herein are incorporated
by reference in their entireties.
* * * * *