U.S. patent application number 10/791303 was filed with the patent office on 2004-12-02 for markers for identification and isolation of pancreatic islet alpha and beta cell progenitors.
Invention is credited to Kritzik, Marcie, Sarvetnick, Nora.
Application Number | 20040241761 10/791303 |
Document ID | / |
Family ID | 22619998 |
Filed Date | 2004-12-02 |
United States Patent
Application |
20040241761 |
Kind Code |
A1 |
Sarvetnick, Nora ; et
al. |
December 2, 2004 |
Markers for identification and isolation of pancreatic islet alpha
and beta cell progenitors
Abstract
The differential expression of marker proteins in a targeted
population provides a means of identifying and isolating cells. A
population of cells associated with the regeneration of pancreatic
islets is shown to express certain proteins, including the cell
surface proteins ErbB2, ErbB3, and ErbB4; and the nuclear protein
Msx-2. Populations of isolated pancreatic islet progenitor cells
find use in screening assays, to characterize genes involved in
islet development and regulation, and in transplantation to provide
a recipient with pancreatic islet functions.
Inventors: |
Sarvetnick, Nora; (San
Diego, CA) ; Kritzik, Marcie; (La Jolla, CA) |
Correspondence
Address: |
BOZICEVIC, FIELD & FRANCIS LLP
1900 UNIVERSITY AVE
SUITE 200
EAST PALO ALTO
CA
94303
US
|
Family ID: |
22619998 |
Appl. No.: |
10/791303 |
Filed: |
March 1, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10791303 |
Mar 1, 2004 |
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09736911 |
Dec 13, 2000 |
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6753153 |
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60170474 |
Dec 13, 1999 |
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Current U.S.
Class: |
435/7.2 |
Current CPC
Class: |
C12Q 1/6881 20130101;
A61P 3/10 20180101; C12N 2503/02 20130101; C07K 14/71 20130101;
C12Q 2600/158 20130101; A61K 35/12 20130101; G01N 33/56966
20130101; A01K 67/0271 20130101; C07K 14/4702 20130101; C12N 5/0678
20130101; C12N 15/8509 20130101; A01K 2217/05 20130101; A01K
2227/105 20130101; C07K 14/705 20130101; Y10T 436/25375 20150115;
A01K 2267/025 20130101; A01K 67/0275 20130101; A01K 2267/0325
20130101 |
Class at
Publication: |
435/007.2 |
International
Class: |
G01N 033/53; G01N
033/567 |
Claims
What is claimed is:
1. A method of identifying mammalian pancreatic islet progenitor
cells, the method comprising: contacting a population of mammalian
pancreatic cells with marker specific binding members for one or
more markers selected from the group consisting of ErbB2, ErbB3,
ErbB4 and Msx-2; and detecting those cells that bind to said marker
specific reagent; wherein cells that bind to said marker specific
reagent are identified as pancreatic islet progenitor cells.
2. The method of claim 1, wherein said pancreatic islet progenitor
cells are progenitors for insulin producing beta cells.
3. The method of claim 2, wherein said marker is ErbB3.
4. The method of claim 2, wherein said marker is Msx-2.
5. The method of claim 2, wherein said method further comprises
contacting said population of pancreatic cells with an
insulin-specific reagent, and detecting those cells that do not
bind to said insulin specific reagent.
6. The method of claim 1, wherein said pancreatic islet progenitor
cells are progenitors for glucagon producing alpha cells.
7. The method of claim 6, wherein said marker is ErbB4.
8. The method of claim 6, wherein said method further comprises
contacting said population of pancreatic cells with a
glucagon-specific reagent, and detecting those cells that do not
bind to said glucagon specific reagent.
9. The method of claim 1, wherein said population of pancreatic
cells are pancreatic duct cells.
10. The method of claim 9, wherein said population of pancreatic
duct cells are from a fetal donor.
11. The method of claim 9, wherein said population of pancreatic
duct cells are from a neonatal donor.
12. The method of claim 9, wherein said population of pancreatic
duct cells are from an adult donor.
13. The method of claim 9, wherein said pancreatic duct cells are
human.
14. The method of claim 9, wherein said pancreatic duct cells are
mouse.
15. The method of claim 1, wherein said marker-specific reagent is
an antibody.
16. The method of claim 15, wherein said antibody comprises a
detectable label.
17. The method of claim 15, wherein said detecting step comprises
detection of said label by flow cytometry.
18. The method of claim 17, further comprising the step of:
separating the cells in said population based on binding to said
marker-specific reagent to provide a purified population of
pancreatic progenitor cells.
19. An isolated population of pancreatic islet progenitor cells,
wherein said cells are derived from pancreatic ducts, and are
characterized as expressing at least one marker selected from the
group consisting ErbB2, ErbB3, ErbB4 and Msx-2.
20. The isolated cell population of claim 19, wherein said
pancreatic islet progenitor cells are progenitors for insulin
producing beta cells.
21. The isolated cell population of claim 20, wherein said marker
is ErbB3.
22. The isolated cell population of claim 21, wherein said cells
are further characterized as lacking detectable production of
insulin.
23. The isolated cell population of claim 19, wherein said
pancreatic islet progenitor cells are progenitors for glucagon
producing alpha cells.
24. The isolated cell population of claim 23, wherein said marker
is ErbB4.
25. The isolated cell population of claim 24, wherein said cells
are further characterized as lacking detectable production of
glucagon.
26. The isolated cell population of claim 19, wherein said cells
are from a fetal donor.
27. The isolated cell population of claim 19, wherein said cells
are from a neonatal donor.
28. The isolated cell population of claim 19, wherein said cells
are from an adult donor.
29. The isolated cell population of claim 19, wherein said cells
are human.
30. The isolated cell population of claim 19, wherein said cells
are mouse.
31. A method of screening for genetic sequences specifically
expressed in pancreatic islet progenitor cells, the method
comprising: isolating RNA from a cell population according to claim
19, generating a probe from said RNA, screening a population of
nucleic acids for hybridization to said probe.
32. The method of claim 31, further comprising a comparison of the
hybridization obtained between said pancreatic islet progenitor
cells and a differentiated cell population.
33. The method of claim 31, wherein said population of nucleic
acids is represented in an array.
34. An in vitro cell culture, comprising: a cell population
according to claim 19; and cell culture medium.
35. A method of screening for agents that affect the growth or
differentiation of pancreatic progenitor cells, the method
comprising: contacting the in vitro culture of claim 34 with a
candidate agent, and determining the effect of said agent on the
growth or differentiation of said pancreatic progenitor cells.
Description
BACKGROUND
[0001] There are 15.7 million people in the United States who have
diabetes, which is the seventh leading cause of death in this
country. As a chronic disease that has no cure, diabetes is one of
the most costly health problems in America. Health care and other
costs directly related to diabetes treatment, as well as the costs
of lost productivity, run $92 billion annually.
[0002] Type I autoimmune diabetes results from the destruction of
insulin producing beta cells in the pancreatic islets of
Langerhans. The adult pancreas has very limited regenerative
potential, and so these islets are not replaced after they are
destroyed. The patient's survival then depends on exogenous
administration of insulin. There are an estimated 500,000 to 1
million people with type 1 diabetes in the United States today. The
risk of developing type 1 diabetes is higher than virtually all
other severe chronic diseases of childhood.
[0003] The optimal treatment of insulin-dependent diabetes mellitus
(IDDM), is the regulated delivery of insulin by functional beta
cells. Pancreas transplantation, however, is a major surgical
procedure with a high rate of complications. Transplantation of the
isolated insulin-secreting islets of Langerhans is an alternative
approach, which is easier and safer than whole organ
transplantation. Clinical trials of islet transplantation have
begun in a few specialized centers worldwide. Insulin independence
at 1 year was achieved in 8% of the patients, but 20% of cases
showed a graft function with a normal basal C peptide and improved
glycemic regulation.
[0004] Beta-cell transplantation has so far been restricted by the
scarcity of human islet donors. This shortage could be alleviated
by methods for the isolation and/or culture of beta-cell
progenitors. Such cells might also be protected from immunological
rejection and recurring autoimmunity by genetic manipulation. The
combination of these approaches with immunoisolation devices holds
the promise of a widely available cell therapy for treatment of
IDDM in the near future.
[0005] The pancreas is composed of at least three types of
differentiated tissue: the hormone-producing cells in islets (4
different cell types), the exocrine zymogen-containing acini, and
the centroacinar cells, ductules and ducts (ductal tree). All of
these cells appear to have a common origin during embryogenesis in
the form of duct-like protodifferentiated cells. Later in life, the
acinar and ductal cells retain a significant proliferative capacity
that can ensure cell renewal and growth, whereas the islet cells
become mitotically inactive.
[0006] During embryonic development, and probably later in life,
pancreatic islets of Langerhans originate from differentiating
epithelial stem cells. These stem cells are situated in the
pancreatic ducts but are otherwise poorly characterized. Pancreatic
islets contain four islet cell types: alpha, beta, delta and
pancreatic polypeptide cells that synthesize glucagon, insulin,
somatostatin and pancreatic polypeptide, respectively. The early
progenitor cells to the pancreatic islets are multipotential and
coactivate all the islet-specific genes from the time they first
appear. As development proceeds, expression of islet-specific
hormones becomes restricted to the pattern of expression
characteristic of mature islet cells.
[0007] The characterization of pre-islet cells is of great interest
for the development of therapeutics to treat diseases of the
pancreas, particularly IDDM. Model systems have been described that
permit the study of these cells. For example, Gu and Sarvetnick
(1993) Development 118:33-46 identify a model system for the study
of pancreatic islet development and regeneration. Transgenic mice
carrying the mouse .gamma.-interferon gene linked to the human
insulin promoter exhibit inflammatory-induced islet loss.
Significant duct cell proliferation occurs in these mice, leading
to a striking expansion of pancreatic ducts. Endocrine progenitor
cells are localized in these ducts. This model provides a source of
progenitor cells for further study.
[0008] The human epidermal growth factor receptor (HER or ErbB)
family consists of four distinct members, including the epidermal
growth factor (EGF) receptor (EGFR, HER1, or ErbB1), ErbB2 (HER2 or
neu), ErbB3 (HER3), and ErbB4 (HER4). Activation of these receptors
plays an important role in the regulation of cell proliferation,
differentiation, and survival in several different tissues. Binding
of a specific ligand to one of the ErbB receptors triggers the
formation of specific receptor homo- and heterodimers, with ErbB2
being the preferred signaling partner.
[0009] The ErbB receptor ligands represent a complex variety of
molecules. The EGF receptor binds six known ligands, including EGF,
TGF.alpha., heparin-binding EGF-like growth factor, amphiregulin,
epiregulin and betacellulin. Other members of the ErbB receptor
family appear to function primarily through interaction with the
neuregulins, a family of EGF-like growth factors encoded by at
least three different genes: NRG1 (NDF, heregulin, GGF, ARIA or
SMDF), NRG2, NRG3 and NRG4. Alternative transcript splicing from
the NRG1 and NRG2 genes results in the production of multiple
neuregulin isoforms. Distinct isoforms can elicit distinct
biological activities depending on the cellular context, thereby
modulating growth and development independently.
[0010] The differential expression of genes by progenitor cells, as
compared to their differentiated progeny, is of interest for the
characterization and isolation of the progenitor cells. Where the
differentially expressed genes encode a receptor for biologically
active molecules, the marker may further provide information about
factors that affect the growth or differentiation of the progenitor
cells. Where such genes encode proteins such as transcription
factors, the marker may provide information about regulated gene
expression in the progenitor cells.
[0011] Relevant Literature
[0012] The expression of ErbB2 in rat embryonic pancreas has been
reported by LeBras et al. (1998) Diabetologia 41:1474-1481. Press
et al. (1990) Oncogene 5:953-962 found that ErbB2 was not
significantly expressed in the adult pancreas, though weak staining
was seen in the ducts. Sundaresan et al. (1998) Endocrinology
139:47564764 have also reported expression of ErbB receptor and
ligand in a ductal epithelial cell derived from rat embryos.
[0013] Hall et al. (1990) J Pathol 161(3):195-200; and Dugan et al.
(1997) Pancreas 14(3):229-36 investigate the expression of ErbB2 in
pancreatic cancers. The receptor has been found to be amplified and
overexpressed in a number of human adenocarcinomas. The data
suggest that there is abnormal expression of c-erb B-2 oncogene in
about 20 percent of cases, although mutational activation was not
seen in human pancreatic adenocarcinoma.
[0014] Oberg-Welsh and Welsh (1996) Pancreas 12:334-339 studied the
expression of protein tyrosine kinases in different preparations of
insulin producing cells by polymerase chain reaction (PCR). Among
the tyrosine kinases thus identified were the fibroblast growth
factor receptor-4 (FGFR-4), c-kit, the insulin-like growth factor
(IGF-1) receptor, and the cytoplasmic tyrosine kinase Jak2, which
associates with the activated receptor for growth hormone (GH).
Fetal islet-like structures were cultured in the absence or
presence of the ligands to these receptors.
[0015] Transcription factors important for insulin gene expression
are critcal for development of the pancreas during embryogenesis
(see Sander and German (1997) J. Mol. Med. 75:327-340). PDX-1 is
one important marker. Like PDX-1, MSX-2 is a homeobox-containing
transcription factor. It is part of a conserved family of
transcription factors that play critical roles in tissue patterning
and organogenesis during development. Msx-2 is expressed at a wide
variety of sites in the developing embryo, but no specific role is
known for pancreatic development (see Davidson (1995) Trends Genet.
11:405-411). It is not expressed in the normal adult pancreas (Maas
et al. (1996) Ann. NY Acad. Sci. 785:171-181).
SUMMARY OF THE INVENTION
[0016] Polypeptide markers are provided that are expressed by
progenitors of pancreatic islet cells. During regeneration of
pancreatic islets, receptors of the ErbB family are expressed,
including ErbB2, ErbB3 and ErbB4. Transcriptional factors are also
shown to be expressed, including the homeobox-containing factor
Msx-2. These markers are useful in identifying progenitor cells in
the lineage that produces pancreatic islet cells; and can also be
used in the detection of pancreatic islet regeneration. The progeny
islet cells include insulin producing beta cells, and glucagon
producing alpha cells.
[0017] Those markers that are expressed on the cell surface are
useful for the enrichment of islet progenitor cells from complex
cell mixtures. Such progenitor populations are useful in
transplantation, for experimental evaluation, and as a source of
lineage and cell specific products, including mRNA species useful
in identifying genes specifically expressed in these cells, and as
targets for the discovery of factors or molecules that can affect
them. Cultures of such cells may utilize ligands that specifically
interact with the cell surface markers. Ligands of interest include
the neuregulins: NRG1, NRG2 and NRG3.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] FIG. 1(A-B). ErbB2 and insulin immunostaining of the fetal
pancreas. Pancreatic sections of an E14.5 embryo were immunostained
with antibody to either ErbB2 (A) or insulin (B) using the ABC
technique (brown is positive staining). Gill's hematoxylin was the
counterstain. ErbB2 is expressed in both the ductal and peri-ductal
regions, while insulin is found mainly in the peri-ductal region.
Original magnifications, 50.times..
[0019] FIG. 2(A-D). ErbB receptor and ligand immunostaining of the
fetal pancreas. Pancreatic sections of an E16 embryo were
immunostained with antibody to either ErbB3 (A), ErbB4 (B), HRG
(C), or HRG 3 (D) using the ABC technique (brown is positive
staining). Gill's hematoxylin was the counterstain. Significant
expression of ErbB receptors and ligands is seen in the fetal
pancreatic ducts. Original magnifications, 40.times..
[0020] FIG. 3(A-C). ErbB receptor and ligand immunostaining of the
IFN.gamma. transgenic pancreas. Pancreatic sections from adult
IFN.gamma. transgenic mice were immunostained with antibody to
either ErbB2 (A), ErbB3 (B), ErbB4 (C), HRG (brown is positive
staining). Gill's hematoxylin was the counterstain. Note the
expression of ErbB receptors and ligands in the pancreatic ducts.
Significant ErbB receptor and ligand expression is not observed in
non-transgenic mice. Original magnifications, 80.times..
[0021] FIG. 4(A-B). Postnatal expression of ErbB2 in the IFN.gamma.
transgenic pancreas. Pancreatic sections from one week (A) or five
week (B) IFN.gamma. transgenic pups were immunostained with
antibody to ErbB2 using the ABC technique (brown is positive
staining). Gill's hematoxylin was the counterstain. There is an
absence of ErbB2 ductal staining in the one week old pup, and
presence of significant ErbB2 immunoreactivity in the five week old
pup. Significant ductal expression of ErbB2 was not observed in
non-transgenic pups at either age. Original magnifications,
80.times. (A); 64.times. (B).
[0022] FIG. 5(A-B). Msx and insulin staining of the fetal pancreas.
Serial sections of an E14.5 embryo were stained with antibody to
either Msx (A) or insulin (B). Magnification 50.times..
[0023] FIG. 6(A-D). Msx immunostaining in the IFN.gamma. transgenic
pancreas. Serial sections of the pancreas from an adult IFN.gamma.
transgenic mouse were stained with antibody to either PDX-1 (A),
Msx (B, C), or insulin (D). Significant expression of Msx is seen,
as well as PDX-1 in panels A and B (the arrows in panel B highlight
Msx-positive cells in the duct wall). Msx-positive cells lacking
insulin expression are indicated by arrows in panel C;
insulin-positive cells lacking Msx expression are indicated by
arrows in panel D. Panels C and D represent serial sections
somewhat offset and at slightly different magnifications.
Magnification A, B, D 40.times.; C 50.times..
[0024] FIGS. 7A and 7B. Engraftment and differentiation of
transplanted in vitro cultured pancreatic ductal "stem cells".
Ductal cells were maintained in culture, and then implanted under
the kidney capsule of an adult IFN.gamma. transgenic. The sections
were stained with antibody to insulin.
DESCRIPTION OF THE SPECIFIC EMBODIMENTS
[0025] A population of cells associated with the regeneration of
pancreatic islets is shown to express certain proteins, including
the cell surface proteins ErbB2, ErbB3, and ErbB4; and the nuclear
protein Msx-2. The differential expression of these proteins in a
progenitor cell population, as compared to the surrounding tissues,
provides a means of identifying and isolating cells that give rise
to pancreatic islet cells, including pancreatic alpha cells and
pancreatic beta cells.
[0026] The progenitor cell population is useful in transplantation
to provide a recipient with pancreatic islet cells, which may
produce insulin or glucagon; for drug screening; experimental
models of islet differentiation and interaction with other cell
types; in vitro screening assays to define growth and
differentiation factors, and to additionally characterize genes
involved in islet development and regulation; and the like. The
native cells may be used for these purposes, or they may be
genetically modified to provide altered capabilities.
[0027] Before the present invention is described, it is to be
understood that this invention is not limited to the particular
embodiments described, as such methods, devices, and formulations
may, of course, vary. It is also to be understood that the
terminology used herein is for the purpose of describing particular
embodiments only, and is not intended to limit the scope of the
present invention which will be limited only by the appended
claims.
[0028] It must be noted that as used herein and in the appended
claims, the singular forms "a," "an," and "the" include plural
referents unless the context clearly dictates otherwise, and
includes reference to equivalent steps and methods known to those
skilled in the art.
[0029] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
any methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, the preferred methods and materials are now described.
All publications mentioned herein are incorporated herein by
reference to disclose and describe the specific methods and/or
materials in connection with which the publications are cited.
[0030] The publications discussed herein are provided solely for
their disclosure prior to the filing date of the present
application. Nothing herein is to be construed as an admission that
the present invention is not entitled to antedate such publication
by virtue of prior invention. Further, the dates of publication
provided may be different from the actual publication dates which
may need to be independently confirmed.
[0031] Definitions
[0032] Markers, as used herein, are nucleic acid or polypeptide
molecules that are differentially expressed in a cell of interest.
In this context, differential expression means an increased level
of the marker for a positive marker, and a decreased level for a
negative marker. The detectable level of the marker nucleic acid or
protein is sufficiently higher or lower in the cells of interest,
compared to other cells, that the cells of interest can be
identified, using any of a variety of methods as known in the
art.
[0033] It will be understood by those of skill in the art that
differential expression is a relative term, and will vary with the
amount of background expression from other cell types. It is also
understood that a marker need not be specifically expressed by a
cell of interest when compared to the body as a whole, as long as
there is specificity within a tissue, organ, or developmental
stage. For example, a protein may be found on a specific cell type
within one organ, but could be widely expressed in other organs, or
at different stages of development. A progenitor cell may
differentially express a polypeptide that is not found in the fully
differentiated progeny cell. A cell of interest may differentially
express a polypeptide that is not expressed in surrounding tissues,
e.g. the subject islet progenitor cells express polypeptides not
found in the normal ductal epithelial cells. This specificity
within an organ is sufficient for purposes of cell identification
and separation.
[0034] Some markers of interest in the present invention include
members of the ErbB family. These molecules, including the
polypeptides and encoding nucleic acids, are well known in the art,
and reagents for the detection thereof are widely available. The
polypeptide and nucleic acid sequence of ErbB2 may be accessed at
Genbank, accession number 004448; ErbB3 at accession number 001982;
and ErbB4 at accession number L07868. Antibodies specific for these
polypeptides are commercially available, or may be produced using
conventional methods as known in the art.
[0035] Another marker of interest is Msx-2, a homeodomain
containing protein. The polypeptide and genetic sequence of Msx-2
may be accessed at Genbank at accession number D31771, and the
protein sequence at accession number BAA06549. See, for example,
Jabs et al. (1993) Cell 75:443-450.
[0036] Pancreatic Cells: Pancreatic tissues, which may be selected
from different developmental stages and sources, are of interest in
the subject invention as a source of islet progenitor cells, and to
supply samples for the further characterization of the islet
development and regeneration process. Depending on the purpose,
whole pancreas may be used, or discrete tissues derived therefrom.
Of particular interest as a source of progenitor cells is
pancreatic ductal tissue, which can be isolated from other
pancreatic tissues by those of skill in the art.
[0037] The subject islet progenitor cells may be isolated from
pancreatic ducts, which may be fetal, neonatal, juvenile or adult.
However, the frequency of progenitor cells is substantially lower
in tissues taken from a host older than a neonate. The progenitor
cells may be obtained from any mammalian species, e.g. equine,
bovine, porcine, canine, feline, rodent, e.g. mice, rats, hamster,
primate, etc., particularly human. The tissue may be obtained by
biopsy from a live donor, or obtained from a dead or dying donor
within about 48 hours of death, or freshly frozen tissue, tissue
frozen within about 12 hours of death and maintained at below about
-20.degree. C., usually at about liquid nitrogen temperature
(-180.degree. C.) indefinitely.
[0038] A tissue source of interest for investigative purposes is
the transgenic mouse described by Gu and Sarvetnick (1993)
Development 118:3346. In such mice, the gene encoding interferon
.gamma. is under the transcriptional control of the insulin
promoter, and therefore is specifically expressed in the pancreas.
In adult life, these animals exhibit significant duct cell
proliferation and islet formation.
[0039] Marker Specific reagent: In a typical assay or separation, a
cell sample is contacted with a marker-specific reagent, and
detecting directly or indirectly the presence of the complex
formed. The term "specific reagent" as used herein refers to a
member of a specific binding pair, i.e. two molecules where one of
the molecules through chemical or physical means specifically binds
to the other molecule. The complementary members of a specific
binding pair are sometimes referred to as a ligand and
receptor.
[0040] In addition to antigen and antibody specific binding pairs,
specific binding members include peptide-MHC antigen and T cell
receptor pairs; complementary nucleotide sequences (including
nucleic acid sequences used as probes and capture agents in DNA
hybridization assays); peptide ligands and receptor, e.g.
neuregulins and ErbB receptors; autologous monoclonal antibodies,
and the like. The specific binding pairs may include analogs,
derivatives and fragments of the original specific binding member.
For example, an antibody directed to a protein antigen may also
recognize peptide fragments, chemically synthesized
peptidomimetics, labeled protein, derivatized protein, etc. so long
as an epitope is present.
[0041] Immunological specific binding pairs include antigens and
antigen specific antibodies or T cell antigen receptors.
Recombinant DNA methods or peptide synthesis may be used to produce
chimeric, truncated, or single chain analogs of either member of
the binding pair, where chimeric proteins may provide mixture(s) or
fragment(s) thereof, or a mixture of an antibody and other specific
binding members. Antibodies and T cell receptors may be monoclonal
or polyclonal, and may be produced by transgenic animals, immunized
animals, immortalized human or animal B-cells, cells transfected
with DNA vectors encoding the antibody or T cell receptor, etc. The
details of the preparation of antibodies and their suitability for
use as specific binding reagents are well-known to those skilled in
the art.
[0042] Of particular interest is the use of antibodies as affinity
reagents. Conveniently, these antibodies are conjugated with a
label for use in separation. Labels include 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.
Fluorochromes that find use include phycobiliproteins, e.g.
phycoerythrin and allophycocyanins, fluorescein and Texas red.
Frequently each antibody is labeled with a different fluorochrome,
to permit independent analysis or sorting for each marker.
[0043] Monoclonal antibodies specific for the subject markers may
be produced in accordance with conventional ways, immunization of a
mammalian host, e.g. mouse, rat, guinea pig, cat, dog, etc., fusion
of resulting splenocytes with a fusion partner for immortalization
and screening for antibodies having the desired affinity to provide
monoclonal antibodies having a particular specificity. These
antibodies can be used for affinity chromatography, ELISA, RIA, and
the like. The antibodies may be labeled with radioisotopes,
enzymes, fluorescers, chemiluminescers, or other label which will
allow for detection of complex formation between the labeled
antibody and its complementary epitope.
[0044] Nucleic acid sequences for detection may be complementary to
the coding or non-coding sequences of the corresponding genes.
Complementary nucleic acids may be cDNA, mRNA or genomic DNA, or a
fragment thereof. Fragments may be obtained of the DNA sequence by
chemically synthesizing oligonucleotides in accordance with
conventional methods, by restriction enzyme digestion, by PCR
amplification, etc. For the most part, DNA fragments will be of at
least 25 nt, usually at least 30 nt, more usually at least about 50
nt. Such small DNA fragments are useful as primers for PCR,
hybridization screening, etc. Where it is desirable to generate
probes or primers that distinguish Msx-2, or an ErbB receptor from
related family members, the probe may be derived from the less
conserved region of the genes.
[0045] For hybridization probes, it may be desirable to use nucleic
acid analogs, in order to improve the stability and binding
affinity. The term "nucleic acid" shall be understood to encompass
such analogs. A number of modifications have been described that
alter the chemistry of the phosphodiester backbone, sugars or
heterocyclic bases. Among useful changes in the backbone chemistry
are phosphorothioates; phosphorodithioates, where both of the
non-bridging oxygens are substituted with sulfur;
phosphoroamidites; alkyl phosphotriesters and boranophosphates.
Achiral phosphate derivatives include 3'-O'-5'-S-phosphorothioate,
3'-S-5'-O-phosphorothioate, 3'-CH.sub.2-5'-O-phosphonate and
3'-NH-5'-O-phosphoroamidate. Peptide nucleic acids replace the
entire phosphodiester backbone with a peptide linkage. Sugar
modifications are also used to enhance stability and affinity. The
.beta.-anomer of deoxyribose may be used, where the base is
inverted with respect to the natural .beta.-anomer. The 2'-OH of
the ribose sugar may be altered to form 2'-O-methyl or 2'-O-allyl
sugars, which provides resistance to degradation without comprising
affinity. Modification of the heterocyclic bases must maintain
proper base pairing. Some useful substitutions include deoxyuridine
for deoxythymidine; 5-methyl-2'-deoxycytidine and
5-bromo-2.quadrature.-deoxycytidine for deoxycytidine.
5-propynyl-2'-deoxyuridine and 5-propynyl-2'-deoxycytidine have
been shown to increase affinity and biological activity when
substituted for deoxythymidine and deoxycytidine, respectively.
Detection of Expression of Markers on Islet Progenitor Cells
[0046] The data provided herein demonstrates that proteins of the
ErbB receptor family, specifically ErbB2, ErbB3 and ErbB4 are
expressed by progenitor cells found in the pancreatic duct, which
cells can develop into pancreatic islet cells. Of particular
utility is ErbB3 as a marker for progenitor cells that go on to
form islet .beta. cells. The expression of ErbB4 is also associated
with progenitor cells that form glucagon producing islet .alpha.
cells. The islet progenitor cells of interest also express the
transcription factor Msx-2.
[0047] Polypeptide Analysis
[0048] Screening may be based on the functional or antigenic
characteristics of the protein, e.g. immunoassays, determination of
Msx-2 directed transcription, response to an ErbB receptor ligand,
etc.
[0049] A pancreatic cell sample, preferably a pancreatic duct
sample, is taken from a suitable donor. Biopsy and autopsy samples
or other sources of pancreatic tissues are of particular interest.
Also included in the invention are derivatives and fractions of
such cells and tissues. The number of cells in a sample will
generally be at least about 10.sup.3, usually at least 10.sup.4,
and may be about 10.sup.5 or more. The cells may be dissociated, in
the case of solid tissues, or tissue sections may be analyzed.
Alternatively a lysate of the cells may be prepared.
[0050] Detection may utilize staining of cells or histological
sections, performed in accordance with conventional methods.
Methods of cell staining may be performed as follows. The
antibodies are added to a suspension of cells, and incubated for a
period of time sufficient to bind the available cell surface
antigens. The incubation will usually be at least about 5 minutes
and usually less than about 30 minutes. It is desirable to have a
sufficient concentration of antibodies in the reaction mixture,
such that the efficiency of the separation is not limited by lack
of antibody. The appropriate concentration is determined by
titration. The medium in which the cells are treated will be any
medium which maintains the viability of the cells. A preferred
medium is phosphate buffered saline containing from 0.1 to 0.5%
BSA. Various media are commercially available and may be used
according to the nature of the cells, including
Dulbecco.quadrature.s Modified Eagle Medium (dMEM), Hank's Basic
Salt Solution (HBSS), Dulbecco.quadrature.s phosphate buffered
saline (dPBS), RPMI, lscove.quadrature.s medium, PBS with 5 mM
EDTA, etc., frequently supplemented with fetal calf serum, BSA,
HSA, etc.
[0051] The antibody may be labeled for direct detection, as
previously described. Alternatively, a second stage antibody or
reagent is used to amplify the signal. Such reagents are well known
in the art. For example, the primary antibody may be conjugated to
biotin, with horseradish peroxidase-conjugated avidin added as a
second stage reagent. Final detection uses a substrate that
undergoes a color change in the presence of the peroxidase. The
absence or presence of antibody binding may be determined by
various methods, including microscopy, radiography, scintillation
counting, etc. The staining intensity of cells can be monitored by
flow cytometry, where lasers detect the quantitative levels of
fluorochrome (which is proportional to the amount of cell surface
antigen bound by the antibodies). Flow cytometry, or FACS, can also
be used to separate cell populations based on the intensity of
antibody staining, as well as other parameters such as cell size
and light scatter. Although the absolute level of staining may
differ with a particular fluorochrome and antibody preparation, the
data can be normalized to a control.
[0052] In order to normalize the distribution to a control, each
cell is recorded as a data point having a particular intensity of
staining. These data points may be displayed according to a log
scale, where the unit of measure is arbitrary staining intensity.
In one example, the brightest cells in a bone marrow sample are
designated as 4 logs more intense than the cells having the lowest
level of staining. When displayed in this manner, it is clear that
the cells falling in the highest log of staining intensity are
bright, while those in the lowest intensity are negative. An
alternative control may utilize a substrate having a defined
density of antigen on its surface, for example a fabricated bead or
cell line, which provides the positive control for intensity.
[0053] An alternative method for diagnosis depends on the in vitro
detection of binding between antibodies and the subject polypeptide
markers in a lysate. Measuring the concentration of marker binding
in a sample or fraction thereof may be accomplished by a variety of
specific assays. A conventional sandwich type assay may be used.
For example, a sandwich assay may first attach marker specific
antibodies to an insoluble surface or support. The particular
manner of binding is not crucial so long as it is compatible with
the reagents and overall methods of the invention. They may be
bound to the plates covalently or non-covalently, preferably
non-covalently. A sample derived from candidate pancreatic islet
progenitor cells is contacted with the bound antigen, and the
presence of bound complexes determined by any convenient
method.
[0054] Other immunoassays are known in the art and may find use.
Ouchterlony plates provide a simple determination of antibody
binding. Western blots may be performed on protein gels or protein
spots on filters, using a detection system specific for marker as
desired, conveniently using a labeling method as described for the
sandwich assay.
[0055] Other diagnostic assays of interest are based on the
functional properties of marker proteins. For example, a functional
assay may be based on the transcriptional changes mediated by Msx-2
gene products. Other assays may, for example, detect DNA
footprinting changes due to complexes formed between Msx-2 and its
binding motif. Ligands to ErbB receptors may be added to a culture
of candidate progenitor cells, and the response measured.
[0056] Nucleic Acid Analysis
[0057] A number of methods are available for analyzing nucleic
acids for the presence or absence of a specific sequence. For
analysis based on nucleic acids, mRNA or nucleic acids derived
therefrom are analyzed for the presence of marker specific
sequences. mRNA in a sample may be used directly, or may be reverse
transcribed to generate a cDNA strand. The cDNA may be amplified by
conventional techniques, such as the polymerase chain reaction
(PCR), to provide sufficient amounts for analysis. The use of the
polymerase chain reaction is described in Saiki, et al. (1985)
Science 239:487, and a review of techniques may be found in
Sambrook, et al. Molecular Cloning: A Laboratory Manual, CSH Press
1989, pp.14.2.quadrature.14.33. Amplification may also be used to
determine whether a specific sequence is present, by using a primer
that will specifically bind to the desired sequence, where the
presence of an amplification product is indicative that a specific
binding complex was formed. Alternatively, the mRNA sample is
fractionated by electrophoresis, e.g. capillary or gel
electrophoresis, transferred to a suitable support, e.g.
nitrocellulose and then probed with a fragment of the marker
sequence. Other techniques may also find use, including
oligonucleotide ligation assays, binding to solid state arrays,
etc. Detection of mRNA having the subject sequence is indicative of
marker gene expression in the sample.
[0058] The sample nucleic acid, e.g. mRNA or amplification product,
is analyzed by one of a number of methods known in the art.
Hybridization with a marker specific sequence may be used to
determine its presence, by in situ hybridization, northern blots,
dot blots, etc. The hybridization pattern of a control and variant
sequence to an array of oligonucleotide probes immobilized on a
solid support, as described in U.S. Pat. No. 5,445,934, or in
WO95/35505, may also be used as a means of detection. For examples
of arrays, see Hacia et al. (1996) Nature Genetics 14:441-447;
Lockhart et al. (1996) Nature Biotechnol. 14:1675-1680; and De Risi
et al. (1996) Nature Genetics 14:457-460.
Isolation of Pancreatic Islet Progenitor Cells
[0059] Methods for enrichment of islet progenitor cell subsets are
provided. The enriched cell population will usually have at least
about 50% cells of the selected phenotype, more usually at least
75% cells of the selected phenotype, and may be 90% or higher of
the selected phenotype. The subject cell populations are separated
from other cells, e.g. differentiated islet and duct cells, on the
basis of specific markers, which are identified with affinity
reagents, e.g. monoclonal antibodies. Markers of interest include
ErbB receptors, as previously described. The separation may also
use negative markers to exclude differentiated epithelial or islet
cells.
[0060] The subject subsets are separated from a complex mixture of
cells by techniques that enrich for cells having the above
characteristics. For isolation of cells from tissue, an appropriate
solution may be used for dispersion or suspension. Such solution
will generally be a balanced salt solution, e.g. normal saline,
PBS, Hank.quadrature.s balanced salt solution, etc., conveniently
supplemented with fetal calf serum or other naturally occurring
factors, in conjunction with an acceptable buffer at low
concentration, generally from 5-25 mM. Convenient buffers include
HEPES, phosphate buffers, lactate buffers, etc.
[0061] Separation of the subject cell populations will then use
affinity separation to provide a substantially pure population.
Techniques for affinity separation may include magnetic separation,
using antibody-coated magnetic beads, affinity chromatography,
cytotoxic agents joined to a monoclonal antibody or used in
conjunction with a monoclonal antibody, e.g. complement and
cytotoxins, and "panning" with antibody attached to a solid matrix,
eg. plate, or other convenient technique. Techniques providing
accurate separation include fluorescence activated cell sorters,
which can have varying degrees of sophistication, such as multiple
color channels, low angle and obtuse light scattering detecting
channels, impedance channels, etc. The cells may be selected
against dead cells by employing dyes associated with dead cells
(e.g. propidium iodide). Any technique may be employed which is not
unduly detrimental to the viability of the selected cells.
[0062] The labeled cells are then separated as to the expression of
one or more of the differentially expressed markers. The separated
cells may be collected in any appropriate medium that maintains the
viability of the cells, usually having a cushion of serum at the
bottom of the collection tube. Various media are commercially
available and may be used according to the nature of the cells,
including dMEM, HBSS, dPBS, RPMI, Iscove.quadrature.s medium, etc.,
frequently supplemented with fetal calf serum.
Culture of Islet Progenitor Cells
[0063] The enriched cell population may be grown in vitro under
various culture conditions. Culture medium may be liquid or
semi-solid, e.g. containing agar, methylcellulose, etc. The cell
population may be conveniently suspended in an appropriate nutrient
medium, such as DMEM: Ham's F12 or RPMI-1640, normally supplemented
with fetal calf serum (about 5-10%), L-glutamine, and antibiotics,
e.g. penicillin and streptomycin.
[0064] The subject progenitor cells are characterized by their
expression of growth factor receptors. In addition to providing a
convenient marker for separation, the cognate ligands may
biologically active on the cells, and find use in the in vitro
culture of the cells. The subject cells express high levels of ErbB
receptors on their cell surface. The biologically relevant receptor
is likely to be a heterodimer between either ErbB2/ErbB3, and/or
ErbB2/ErbB4.
[0065] The culture may contain growth factors to which the cells
are responsive. Growth factors, as defined herein, are molecules
capable of promoting survival, growth and/or differentiation of
cells, either in culture or in the intact tissue, through specific
effects on a transmembrane receptor. Growth factors include
polypeptides and non-polypeptide factors. Specific growth factors
that may be used in culturing the subject cells include heregulin,
epidermal growth factor, TGF.alpha., heparin-binding EGF-like
growth factor, keratinocyte growth factor, amphiregulin,
epiregulin, betacellulin, NRG2, NRG3, NRG4, and the like. The
specific culture conditions are chosen to achieve a particular
purpose, i.e. differentiation into insulin producing cell
populations, maintenance of progenitor cell activity, etc.
[0066] The subject cells are found to grow as a monolayer. However,
in some circumstances it may be desirable to include a co-culture
with stromal or feeder layer cells. Stromal cells suitable for use
in the growth of hematopoietic cells are known in the art. These
include fibroblasts, e.g. pancreatic derived fibroblasts, STO cells
(Axelrod (1984) Dev Biol 101(1):225-8; Kitani et al. (1996) Zoolog
Sci 13(6):865-71); bone marrow stroma as used in "Whitlock-Witte"
(Whitlock et al. [1985] Annu Rev Immunol 3:213-235) or "Dexter"
culture conditions (Dexter et al. [1977] J Exp Med 145:1612-1616);
and heterogeneous thymic stromal cells (Small and Weissman [1996]
Scand J Immunol 44:115-121).
Uses of Progenitor Cells
[0067] The subject cultured cells may be used in a wide variety of
ways. The nutrient medium, which is a conditioned medium, may be
isolated at various stages and the components analyzed. Separation
can be achieved with HPLC, reversed phase-HPLC, gel
electrophoresis, isoelectric focusing, dialysis, or other
non-degradative techniques, which allow for separation by molecular
weight, molecular volume, charge, combinations thereof, or the
like. One or more of these techniques may be combined to enrich
further for specific fractions.
[0068] The progenitor cells may be used in conjunction with the
culture system in the isolation and evaluation of factors
associated with the differentiation and maturation of islet cells.
Thus, the progenitor cells may be used in assays to determine the
activity of media, such as conditioned media, evaluate fluids for
growth factor activity, involvement with dedication of lineages, or
the like.
[0069] The subject islet progenitor cell populations may be used
for reconstitution of islet cell function in a recipient, e.g.
insulin producing beta cells, glucagon producing cells, etc. The
condition may be caused by genetic or environmental conditions,
e.g. autoimmune diseases, type I diabetes mellitus, etc. Autologous
cells or allogeneic cells, may be used for progenitor cell
isolation and subsequent transplantation.
[0070] Genes may be introduced into the progenitor cells for a
variety of purposes, e.g. prevent HIV infection, replace genes
having a loss of function mutation, etc. Alternatively, vectors are
introduced that express antisense mRNA or ribozymes, thereby
blocking expression of an undesired gene. Other methods of gene
therapy are the introduction of drug resistance genes to enable
normal progenitor cells to have an advantage and be subject to
selective pressure, for example the multiple drug resistance gene
(MDR), or anti-apoptosis genes, such as bcl-2. Various techniques
known in the art may be used to transfect the target cells, e.g.
electroporation, calcium precipitated DNA, fusion, transfection,
lipofection and the like. The particular manner in which the DNA is
introduced is not critical to the practice of the invention.
[0071] Many vectors useful for transferring exogenous genes into
target mammalian cells are available. The vectors may be episomal,
e.g. plasmids, virus derived vectors such cytomegalovirus,
adenovirus, etc., or may be integrated into the target cell genome,
through homologous recombination or random integration, e.g.
retrovirus derived vectors such MMLV, HIV-1, ALV, etc. For examples
of progenitor and stem cell genetic alteration, see Svendsen et al.
(1999) Trends Neurosci. 22(8):357-64; Krawetz et al. (1999) Gene
234(1):1-9; Pellegrini et al. Med Biol Eng Comput. 36(6):778-90;
and Alison (1998) Curr Opin Cell Biol. 10(6):710-5.
[0072] To prove that one has genetically modified progenitor cells,
various techniques may be employed. The genome of the cells may be
restricted and used with or without amplification. The polymerase
chain reaction; gel electrophoresis; restriction analysis;
Southern, Northern, and Western blots; sequencing; or the like, may
all be employed. The cells may be grown under various conditions to
ensure that the cells are capable of differentiation while
maintaining the ability to express the introduced DNA. Various
tests in vitro and in vivo may be employed to ensure that the
pluripotent capability of the cells has been maintained.
[0073] The progenitor cells may be administered in any
physiologically acceptable medium, e.g. intravascularly, including
intravenous, although they may also be introduced into other
convenient sites, where the cells may find an appropriate site for
regeneration and differentiation. Usually, at least
1.times.10.sup.3 cells will be administered, more usually at least
about 1.times.10.sup.4, preferably 1.times.10.sup.6 or more. The
cells may be introduced by injection, catheter, or the like.
[0074] 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
progenitor cells in vivo and are, therefore, the preferred form in
which the progenitor cells are introduced into the recipient
subjects.
[0075] Some treatments for diabetes have utilized the
transplantation of healthy pancreatic islets, usually encapsulated
in a membrane to avoid immune rejection. For example, a tubular
membrane can be coiled in a housing that contained islets. The
membrane is connected to a polymer 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 can be maintained (Sullivan et al.
(1991) Science 252:718). Alternatively, hollow fibers containing
islet cells can be immobilized in a polysaccharide alginate (Lacey
et al. (1991) Science 254:1782). Islets have also been placed in
microcapsules composed of alginate or polyacrylates.
[0076] 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.
[0077] Many methods for encapsulating cells are known in the art.
For example, insulin producing cells or progenitors thereof may be
encapsulated in implantable hollow fibers. Such fibers can be
pre-spun and subsequently loaded with the cells, or can be
co-extruded with a polymer which acts to form a polymeric coat
about the cells.
[0078] 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.
[0079] The subject cells are useful for in vitro assays and
screening to detect factors that are active on islet progenitors. A
wide variety of assays may be used for this purpose, including
immunoassays for protein binding; determination of cell growth,
differentiation and functional activity; production of hormones;
and the like.
[0080] Of particular interest is the examination of gene expression
in the progenitor cells of the invention. The expressed set of
genes may be compared with a variety of cells of interest, e.g.
insulin producing beta cells, fetal pancreatic tissues, etc., as
known in the art. For example, in order to determine the genes that
are regulated during development, one could compare the set of
genes expressed by duct cells to islet cells.
[0081] Any suitable qualitative or quantitative methods known in
the art for detecting specific mRNAs can be used. mRNA can be
detected by, for example, hybridization to a microarray, in situ
hybridization in tissue sections, by reverse transcriptase-PCR, or
in Northern blots containing poly A+ mRNA. One of skill in the art
can readily use these methods to determine differences in the size
or amount of mRNA transcripts between two samples. For example, the
level of particular mRNAs in progenitor cells is compared with the
expression of the mRNAs in a reference sample, e.g. differentiated
cells.
[0082] Any suitable method for detecting and comparing mRNA
expression levels in a sample can be used in connection with the
methods of the invention. For example, mRNA expression levels in a
sample can be determined by generation of a library of expressed
sequence tags (ESTs) from a sample. Enumeration of the relative
representation of ESTs within the library can be used to
approximate the relative representation of a gene transcript within
the starting sample. The results of EST analysis of a test sample
can then be compared to EST analysis of a reference sample to
determine the relative expression levels of a selected
polynucleotide, particularly a polynucleotide corresponding to one
or more of the differentially expressed genes described herein.
[0083] Alternatively, gene expression in a test sample can be
performed using serial analysis of gene expression (SAGE)
methodology (Velculescu et al., Science (1995) 270:484). In short,
SAGE involves the isolation of short unique sequence tags from a
specific location within each transcript. The sequence tags are
concatenated, cloned, and sequenced. The frequency of particular
transcripts within the starting sample is reflected by the number
of times the associated sequence tag is encountered with the
sequence population.
[0084] Gene expression in a test sample can also be analyzed using
differential display (DD) methodology. In DD, fragments defined by
specific sequence delimiters (e.g., restriction enzyme sites) are
used as unique identifiers of genes, coupled with information about
fragment length or fragment location within the expressed gene. The
relative representation of an expressed gene with a sample can then
be estimated based on the relative representation of the fragment
associated with that gene within the pool of all possible
fragments. Methods and compositions for carrying out DD are well
known in the art, see, e.g., U.S. Pat. No. 5,776,683; and U.S. Pat.
No. 5,807,680.
[0085] Alternatively, gene expression in a sample using
hybridization analysis, which is based on the specificity of
nucleotide interactions. Oligonucleotides or cDNA can be used to
selectively identify or capture DNA or RNA of specific sequence
composition, and the amount of RNA or cDNA hybridized to a known
capture sequence determined qualitatively or quantitatively, to
provide information about the relative representation of a
particular message within the pool of cellular messages in a
sample. Hybridization analysis can be designed to allow for
concurrent screening of the relative expression of hundreds to
thousands of genes by using, for example, array-based technologies
having high density formats, including filters, microscope slides,
or microchips, or solution-based technologies that use
spectroscopic analysis (e.g., mass spectrometry). One exemplary use
of arrays in the diagnostic methods of the invention is described
below in more detail.
[0086] Hybridization to arrays may be performed, where the arrays
can be produced according to any suitable methods known in the art.
For example, methods of producing large arrays of oligonucleotides
are described in U.S. Pat. No. 5,134,854, and U.S. Pat. No.
5,445,934 using light-directed synthesis techniques. Using a
computer controlled system, a heterogeneous array of monomers is
converted, through simultaneous coupling at a number of reaction
sites, into a heterogeneous array of polymers. Alternatively,
microarrays are generated by deposition of pre-synthesized
oligonucleotides onto a solid substrate, for example as described
in PCT published application no. WO 95/35505.
EXPERIMENTAL
[0087] The following examples are put forth so as to provide those
of ordinary skill in the art with a complete disclosure and
description of how to make and use the subject invention, and are
not intended to limit the scope of what is regarded as the
invention. Efforts have been made to ensure accuracy with respect
to the numbers used (e.g. amounts, temperature, concentrations,
etc.) but some experimental errors and deviations should be allowed
for. Unless otherwise indicated, parts are parts by weight,
molecular weight is average molecular weight, temperature is in
degrees centigrade; and pressure is at or near atmospheric.
[0088] It is to be understood that this invention is not limited to
the particular methodology, protocols, cell lines, animal species
or genera, and reagents described, as such may vary. It is also to
be understood that the terminology used herein is for the purpose
of describing particular embodiments only, and is not intended to
limit the scope of the present invention which will be limited only
by the appended claims.
[0089] As used herein the singular forms "a", "and", and "the"
include plural referents unless the context clearly dictates
otherwise. Thus, for example, reference to "a cell" includes a
plurality of such cells and reference to "the protein" includes
reference to one or more proteins and equivalents thereof known to
those skilled in the art, and so forth. All technical and
scientific terms used herein have the same meaning as commonly
understood to one of ordinary skill in the art to which this
invention belongs unless clearly indicated otherwise.
Example 1
Expression of ErbB Receptors During Pancreatic Islet Development
and Regrowth
[0090] Expression of the ErbB receptor family and one of its
ligands, heregulin, were characterized in order to identify
molecules associated with pancreatic development and regeneration.
In addition to studying expression during fetal pancreatic
development, the expression was studied during pancreatic
regeneration in the IFN.gamma. transgenic mouse, which exhibits
significant duct cell proliferation and new islet formation. These
studies demonstrate significant expression of the ErbB2, ErbB3, and
ErbB4 receptors, as well as heregulin isoforms, in the developing
fetal pancreas.
[0091] In addition, significant ductal expression of these proteins
was found during IFN.gamma.-mediated pancreatic regeneration. This
striking expression was largely absent in one week old neonates but
was clearly visible in pups by five weeks of age. These data
therefore indicate that ErbB receptor and ligand expression decline
by birth in both the IFN.gamma. transgenic and non-transgenic mice,
and that expression resumes early in postnatal life in the
IFN.gamma. transgenic mice. The expression of ErbB receptor family
members at sites of islet development and regrowth suggests that
these molecules are relevant to these processes.
[0092] Materials and Methods
[0093] Animal Husbandry. Animals were maintained in a specific
pathogen-free facility at The Scripps Research Institute according
the rules and regulations governed and enforced by the
Institutional Animal Care and Use Committee. Animals were housed
under a controlled 12-hour light/dark cycle and provided with food
and water ad libitum.
[0094] Transgenic Mice. Transgenic mice expressing IFN.gamma. have
been described previously (Gu et al. (1993), supra.) The IFN.gamma.
transgenic mice used in these studies were on the NOD background.
IFN.gamma. mice that have been backcrossed onto the NOD/Shi strain
for more than ten generations have a very low incidence of
diabetes, <20% as compared to NOD mice, which exhibit an
incidence of .about.25% for males and .about.80% for females.
Embryos were harvested at defined stages of development, with the
day of plug formation counted as day 0.5.
[0095] Immunohistochemistry. Pancreata harvested from mice were
fixed overnight in 10% neutral buffered formalin (3.6%
formaldehyde) and embedded in paraffin. 5 m paraffin sections were
either conventionally stained with hematoxylin and eosin (H&E)
for histological evaluation or were stained with antibodies against
ErbB2, ErbB3, ErbB4, HRG or HRG 3 using immunocytochemical
techniques. Briefly, sections were deparaffinized and blocked with
2% normal goat serum before applying the primary antibodies (Santa
Cruz Biotechnology, Santa Cruz, CA: #sc-284 (ErbB2); #sc-285
(ErbB3); #sc-283 (ErbB4); #sc-348 (HRG); #sc-347 (HRG 3)). These
antibodies were raised against peptides (17-20 amino acids in
length) specific for each protein. As reported by the manufacturer,
the antibodies against ErbB2, ErbB3, and ErbB4 do not cross react
in Western blots of murine samples. We see distinct patterns of
expression with each of these antibodies in immunohistochemistry as
well, further indicating the specificity of these reagents. In
addition, the HRG 3 antibody does not cross react with other
heregulin family members. The HRG antibody is directed against a
peptide from the carboxyl terminus of the heregulin precursor.
Although it is non cross-reactive with mature forms of the
heregulin family and will not recognize HRG 3, other alpha and beta
heregulin isoforms might be detected with this reagent. Binding of
the primary antibody was detected using the appropriate secondary
antibody (Vector Laboratories, Burlingame, Calif.), and the
horseradish peroxidase (HRP)-labeled avidin-biotin complex (ABC
kit, Vector Laboratories). Endogenous peroxidase activity was
quenched with 1% hydrogen peroxide in methanol. HRP was visualized
using 3,3'-diaminobenzidine as a substrate. Gill's hematoxylin was
used as a counterstain.
[0096] Results
[0097] ErbB receptor and ligand expression during fetal
development. To begin to assess the involvement of ErbB receptors
during pancreatic development, we first sought to characterize ErbB
receptor expression in the fetal pancreas during mouse development.
We have previously demonstrated the presence of ErbB1 (the EGF
receptor) in the apical cytoplasm of select acinii in the
IFN.gamma. transgenic pancreas. These acinii appeared to be in a
state of transition, as they exhibited ductal characteristics. Most
ducts in the transgenic pancreas did not express ErbB1. Therefore,
we chose to concentrate our studies on the other members of the
ErbB receptor family.
[0098] To this end, fetal sections taken from non-transgenic mice
were analyzed for expression of the ErbB2, ErbB3, and ErbB4
receptors. As indicated in FIG. 1A, we observed significant
expression of ErbB2 in the primitive ducts of the fetal pancreas at
E14.5 (FIG. 1A). We also observed some staining in cells within
acinar structures. In comparison with ErbB2 receptor staining
patterns, FIG. 1B illustrates the pattern of insulin staining in
the E14.5 fetal pancreas. As illustrated in this Figure, most of
the insulin stain is localized to the periphery of the fetal ducts.
As with ErbB2 staining, ErbB3 and Erb4 were also expressed in the
fetal ducts during pancreatic development (FIGS. 2A and 2B).
[0099] Given the expression of the ErbB receptors in the fetal
pancreas, we anticipated that ErbB receptor ligands would also be
present in this region. The expression of heregulin isoforms was
also assessed. These analyses demonstrated significant
immunoreactivity with both the HRG and HRG 3 antibodies in the
fetal ducts, indicating the presence of heregulin isoforms during
fetal pancreatic development (FIGS. 2C and 2D). The demonstration
of significant ErbB receptor and ligand expression in the primitive
ducts of the fetal pancreas is striking and, given the ductal
derivation of endocrine cells during pancreatic development, it
suggests that this receptor family is important for pancreatic
islet development during ontogeny.
[0100] ErbB receptor expression in pancreatic ducts of the IFNg
transgenic pancreas immunohistochemistry was used to define ErbB
receptor expression patterns during regeneration in the pancreatic
ducts of the IFN.gamma. transgenic mouse. FIGS. 3A, 3B, and 3C
illustrate ErbB2, ErbB3, and ErbB4 receptor staining in the
IFN.gamma. transgenic pancreas, respectively. Significantly, we
observed substantial expression of ErbB receptors in duct cells of
the regenerating pancreas. The extent and proportion of ErbB
receptor-expressing cells varied among ducts, with some ducts
containing few, if any, ErbB positive cells, while other ducts
contained many ErbB positive cells. In general, ErbB2 staining was
much more widespread than ErbB3 or ErbB4 staining. Although the
intense ErbB receptor expression patterns observed in ducts of the
IFN.gamma. transgenic pancreas were not observed in the
non-transgenic pancreas, we did observe very occasional weak ErbB
receptor expression in the ducts of non-transgenic mice. We also
stained the regenerating pancreas for expression of heregulin
isoforms. We observed significant duct cell-specific expression
using both the HRG and HRG 3 antibodies in the transgenic pancreas
as well. The acinar tissue did not stain with any of the heregulin
ligand or receptor antibodies used.
[0101] ErbB receptor and ligand expression in pancreatic ducts
during postnatal pancreatic development. The expression of ErbB
receptors in the fetal pancreas as well as in the IFN.gamma.
transgenic pancreas is in contrast to our observations in the adult
non-transgenic pancreas. These findings suggested that either ErbB
receptor expression was maintained in the transgenic pancreas from
early in development through adulthood, or that it declined as in
the non-transgenic fetal pancreas, only to be induced postnatally.
These distinct possibilities prompted us to examine the pattern of
ErbB receptor expression in the IFN.gamma. transgenic and
non-transgenic pancreas early in postnatal life.
[0102] We stained pancreatic sections taken from IFN.gamma.
transgenic and non-transgenic mice soon after birth, at one week of
age, with the ErbB2, ErbB3, ErbB4, HRG, and HRG 3 antibodies.
Interestingly, we did not detect significant staining in the ducts
of the transgenic or non-transgenic mice with any of the antibodies
screened (FIG. 4A). We next examined expression a few weeks later,
at five weeks of age, in order to determine if expression had
recommenced in the transgenic mice at this point. At this age, we
were already able to observe expansion of pancreatic ducts in the
transgenic mice as compared to the non-transgenic mice. Indeed, by
this time, we did observe significant ductal staining using the
ErbB receptor and ligand antibodies in the IFN.gamma. transgenic
mice, but not in the non-transgenic mice (FIG. 4B). These data
therefore indicate that while ErbB receptor and ligand expression
decline significantly by birth in both IFN.gamma. transgenic and
non-transgenic mice, expression resumes in the transgenic mice
relatively early in postnatal life.
[0103] ErbB receptor expression in pancreatic islets. We observed a
striking induction of intra-islet ErbB2 expression in islets that
exhibited infiltration by inflammatory cells, compared to
non-infiltrated islets (FIGS. 5A and 5B). Within the same field, we
observed islets exhibiting different degrees of ErbB2 induction,
with the degree of expression correlated with the extent of
lymphocytic infiltration of the islet. Indeed, the enhanced ErbB2
islet staining was found to be adjacent to the infiltrates. This
striking pattern of staining was not observed in the absence of
islet infiltration, as in NOD.SCID mice. ErbB4 receptor staining
also exhibited a distinct pattern of expression above the basal
level of staining previously described. That is, elevated ErbB4
expression was predominantly localized at the periphery of the
islets, in a pattern reminiscent of glucagon-secreting A cell
organization (FIGS. 5C and 5D). This pattern of expression is
distinct from insulin staining patterns. In contrast, islet
staining with the ErbB3 antibody did not exhibit such striking
patterns of expression.
[0104] The ErbB receptor family members are expressed at a wide
variety of sites during development, including the fetal pancreas.
In these studies, we have examined the expression of this receptor
family during fetal pancreatic development, where we have observed
defined expression of ErbB receptors in the primitive fetal ducts,
from which endocrine cells and hence new islets develop. In
addition, we have explored receptor expression in the IFN.gamma.
transgenic mouse, which exhibits striking duct cell proliferation
and new islet formation throughout adulthood. The expression of
ErbB receptor family members during new islet formation in the
IFN.gamma. transgenic mouse, as well as in islet development during
ontogeny, suggests a role for these molecules in the development
and regrowth of pancreatic islets.
[0105] The ductal derivation of endocrine cells during fetal islet
development has been well described, and it has been previously
shown that endocrine cells derive from duct cells in the IFN.gamma.
transgenic pancreas (Gu et al. (1994) supra.) These data suggest
that endocrine progenitor cells exist within the pancreatic ducts
and can initiate new islet growth under the appropriate conditions.
Given their expression during islet growth in development (in the
fetal pancreas) and regeneration (in the IFN.gamma. transgenic
pancreas), our data further suggest that the ErbB receptors are
associated with endocrine progenitor cells within the duct
wall.
[0106] Whereas ducts in the IFN.gamma. transgenic pancreas
exhibited significant expression of the ErbB receptors and the
neuregulins, those in the non-transgenic NOD pancreas did not. This
is in contrast to studies of the fetal pancreas during development.
Upon examination of ErbB receptor and ligand staining patterns
during pancreatic development, considerable staining was seen in
the fetal ducts of non-transgenic embryos. In addition, ductal
expression of these receptors diminishes by one week of age,
although it is resumed in the pancreatic ducts after a few weeks of
life in the IFN.gamma. transgenic mice.
[0107] Considerable evidence exists which suggests that the
formation of receptor heterodimers between members of the ErbB
receptor family play an important role in mediating signaling
events. Indeed, although nearly all receptor combinations are able
to form, ErbB2 appears to be the preferred heterodimerization
partner for the other ErbB receptors. In addition, receptor
complexes with ErbB2 appear to have the highest affinity for ligand
and are the most active complexes. However, none of the ligands
described so far appears to bind ErbB2 directly. Rather, a variety
of studies have shown that several ligands, including EGF,
neuregulins, and betacellulin, are able to mediate the
phosphorylation and activation of ErbB2, via heterodimerization
with ErbB1, ErbB3, or ErbB4. Recent work has demonstrated that the
ability of cells to respond to a given ligand is dependent on the
expression of specific ErbB receptor combinations in the target
tissue. In addition, Jones et al. have recently documented extreme
variations in ligand binding specificities and affinities observed
with different ErbB receptor dimer combinations (Jones et al.
(1999) FEBS 447 227-231). Thus diversity in signaling events
involving ErbB receptors is generated by a number of critical
factors, including ligand isoforms, receptor dimer composition, and
the restricted expression of receptors during development and in
adulthood.
[0108] The expression of ErbB2, ErbB3, and ErbB4 receptors in the
IFN.gamma. transgenic pancreas suggests that multiple combinations
of receptor heterodimers are able to form, enabling interaction
with a wide range of ligands and generating diversity in signaling
events. The detection of heregulin family members at times of ErbB
receptor expression suggests that these molecules, which have a
high affinity for the ErbB2/3 and ErbB2/4 receptor heterodimers,
are likewise involved in ErbB receptor-mediated events.
[0109] We observed mainly membrane-associated and cytoplasmic
staining of the ErbB receptors. In addition, we have also observed
some expression over the nuclear region in the fetal as well as in
the IFN.gamma. transgenic pancreas. This was particularly evident
for ErbB3, ErbB4, and HRG in the acinar and ductal regions of the
fetal pancreas. Furthermore, the occasional duct staining that was
observed in the non-transgenic pancreas with the ErbB2, ErbB3, and
ErbB4 antibodies was often over the nuclear region; no cytoplasmic
or membrane-associated ErbB receptor staining was detected in these
samples.
[0110] There was a significant enhancement of ErbB2 expression in
islets that correlated with the extent of lymphocytic infiltration
of the islet. This increase in the expression of ErbB2 was
striking, as intact islets adjacent to infiltrated islets in the
same section did not display such increased expression of ErbB2.
Interestingly, ErbB4 gave a distinct pattern of islet expression as
well. In this case, the peripherally localized expression of ErbB4
in the pancreatic islets was reminiscent of that seen for the
glucagon-expressing A cells, and it suggests that ErbB4 might
participate directly in the differentiation and/or maintenance of
this endocrine cell type.
[0111] In summary, the above results demonstrate significant
expression of the ErbB2, ErbB3, and ErbB4 receptor tyrosine
kinases, as well as heregulin ligands, in the fetal pancreas and in
the IFN.gamma. transgenic pancreas. The association of these
molecules with endocrine cell and islet formation suggests that
they play important roles in mediating new islet growth during
pancreatic development, as well as during IFN.gamma.-mediated
pancreatic regeneration.
Example 2
Expression of Msx-2 in the Regenerating and Developing Pancreas
[0112] Elevated expression of the homeobox-containing protein Msx-2
was observed in the pancreata of fetal mice as well as adult
IFN.gamma. mice, identifying this molecule as a novel marker
associated with pancreatic development and regeneration as well.
The identification of PDX-1 and Msx in the ducts of the IFN.gamma.
transgenic pancreas but not in ducts of the non-transgenic pancreas
suggests that these molecules are associated with endocrine
precursor cells in the ducts of the IFN.gamma. transgenic
mouse.
[0113] Transcription factors important for insulin gene expression
are critical for the development of the pancreas during
embryogenesis (see Sander and German (1997) J Mol Med 75 327-340).
PDX-1 (also called IDX-1, IPF-1, or STF-1), a transcription factor
that regulates insulin expression, is one important marker. Like
PDX-1, Msx-2 is a homeobox-containing transcription factor. Msx-2
is part of a conserved family of transcription factors that play
critical roles in tissue patterning and organogenesis during
development (Davidson and Hill (1991), supra.) For example, the
involvement of Msx-2 in bone and tooth development has been
well-described. Notably, Msx-2 is expressed at a wide variety of
sites in the developing embryo, suggesting its involvement in the
generation of a number of organ systems. Nevertheless, no specific
role is known for Msx-2 in pancreatic development.
[0114] Materials and Methods
[0115] Animal husbandry. Animals were maintained in a specific
pathogen-free facility at The Scripps Research Institute according
the rules and regulations governed and enforced by the
Institutional Animal Care and Use Committee. Animals were housed
under a controlled 12-hour light/dark cycle and provided with food
and water ad libitum. The embryos used in these studies did not
carry the IFN.gamma. transgene.
[0116] Transgenic mouse generation. Transgenic mice expressing
IFN.gamma. have been described previously (Sarvetnick et al. (1988)
supra.) The IFN.gamma. transgenic mice used in these studies were
on the NOD background. IFN.gamma. mice that have been backcrossed
onto the NOD/Shi strain for more than ten generations have a very
low incidence of diabetes, <20%, compared to NOD mice, which
have an incidence of .about.80% for females and .about.25% for
males.
[0117] Immunohistochemistry. Pancreata from test mice were fixed
overnight in 10% neutral buffered formalin (3.6% formaldehyde) and
embedded in paraffin. 5 m paraffin sections were either
conventionally stained with hematoxylin and eosin (H&E) for
histological evaluation or stained for the presence of insulin,
PDX-1, or Msx using immunocytochemical techniques. Briefly,
sections were deparaffinized and blocked with 2% normal goat serum
before applying the primary antibodies for insulin (DAKO,
Carpentaria, Calif.), Msx (BAbCO, Richmond, Calif.), or PDX-1 (a
generous gift from Dr. Chris Wright, Vanderbilt University Medical
School, Nashville, Tenn. and Dr. Helena Edlund, University of
Ume{dot over (a)}, Ume{dot over (a)}, Sweden). Binding of the
primary antibody was detected using the appropriate secondary
antibody (Vector Laboratories, Burlingame, Calif.), and the
horseradish peroxidase (HRP)-labeled avidin-biotin complex (ABC
kit, Vector Laboratories). HRP was visualized using
3,3'-diaminobenzidine as a substrate. Gill's hematoxylin was used
as a counterstain.
[0118] Immunoelectron Microscopy. Pancreatic tissue was fixed in
10% normal buffered formalin (3.6% formaldehyde) for 2 hours at
25.degree. C. Fixed tissue was infused in 1.5M sucrose-PBS for 0.5
hours with gentle inversion periodically. Infused tissue was then
quick-frozen in liquid nitrogen, embedded in OCT and 2-methylbutane
and sectioned 30-40 m thick. These sections were incubated in
glycine-PBS to quench aldehyde for 0.5 hours, blocked in 10% normal
goat serum for 0.5 hours, incubated for 1 hour each in PDX-1
(primary antibody) and an HRP-conjugated goat anti-rabbit secondary
antibody before refixing in 1% glutaraldehyde-PBS for 0.25 hours
and washing in PBS. The reaction product was visualized with
diaminobenzidine (DAB) for 7 minutes and DAB+H.sub.2O.sub.2 for 4
minutes before treating with 1% OsO.sub.4. Tissue was dehydrated in
graded EtOH, cleared in propylene oxide and embedded in Spurr
resin. Thin sections were viewed with a Hitachi HU 12A electron
microscope.
[0119] Differential Gene Expression Analysis. The Atlas Mouse cDNA
Expression Array I (Clontech, Palo Alto, Calif.) was used to screen
the IFN.gamma. NOD-SCID-regenerating pancreas for upregulation of
mRNAs relative to the non-transgenic NOD-SCID pancreas. The
analysis was carried out according to the manufacturer's
recommendations. Msx-2 was one of nineteen transcripts found to be
expressed in the regenerating pancreas but not in the
non-transgenic pancreas.
[0120] Results
[0121] PDX-1 and Msx in the fetalpancreas. We first sought to
characterize PDX-1, Msx-2, and insulin expression during fetal
pancreatic development for comparison to that during regeneration,
with the results summarized in Table 1. After staining of pancreata
from E14.5 Balb/c embryos, PDX-1 reactivity was most notable in the
cord region of expanding epithelial tissue from which the ducts and
endocrine tissue develop, and was also observed in the acinar
tissue, consistent with previous reports. Although less extensive
than PDX-1 staining, Msx displayed considerable staining in the
expanding epithelia of the developing pancreas where PDX-1 was also
detected. Both nuclear and diffuse cytoplasmic staining were
observed with this antibody as well, and faint nuclear staining in
the acinar tissue was also detected (FIG. 6A). Finally,
insulin-producing cells were also found in the region of expanding
epithelia in the embryonic pancreas (FIG. 6B). Compared to PDX-1
and Msx staining, its expression was not as widespread within the
cords, but rather was restricted more to the peri-epithelial region
(FIG. 6 illustrates this point in a comparison of Msx and insulin
staining patterns).
1TABLE 1 Summary of PDX-1, Msx and Insulin Staining PDX-1 MSX
Insulin Fetal Balb/c duct (cord) +++ + +/- peri-epithelial +++ ++
++ acinar ++ +/- - Adult IFN.gamma. transgenic duct +++ ++ + acinar
- - - islet +++ + +++ non-transgenic duct - - - acinar - - - islet
+++ + +++ Plus signs refer to the extent of staining, from low
(+/-_to extensive (+++). Peri-epithelial staining refers to
staining abutting the ductal cord region.
[0122] Msx expression in IFNg mice. Sections of regenerating
pancreata from adult IFNg mice were screened for the presence of
the Msx proteins by staining with a polyclonal antibody directed
against the Msx homeodomain. The only antibody available for this
purpose recognizes both the Msx-1 and Msx-2 proteins and probably
Msx-3 as well. Our analyses demonstrated diffuse Msx staining
throughout the islets of all mice assessed. In contrast, acinar
tissue did not stain with the Msx antibody. Importantly, a
significant portion of duct cells in the regenerating pancreas were
positive for Msx expression (FIGS. 7B and 7C). We observed staining
in the nucleus, as expected for members of this transcription
factor family; however, cytoplasmic staining was also observed in
many instances. The extent of staining varied among ducts; some
ducts had many positive cells and other ducts had few, if any,
Msx-positive cells. No Msx expression was evident in the ducts of
non-transgenic control mice. Furthermore, Msx staining was less
extensive than PDX-1 staining in the transgenic ducts, and
comparison of serial section staining patterns suggested that
Msx-expressing cells also expressed PDX-1 (FIGS. 7A and 7B).
Subsequent analyses further suggested coincident staining between
insulin and Msx in many instances.
[0123] Although the similarity in Msx and insulin expression
patterns could reflect a basal level of cross-reactivity between
the Msx antibodies and endocrine cells (supported by the diffuse
islet staining we observe with the Msx antibody), we identified
Msx-positive cells that did not stain for insulin in a number of
ducts; insulin-positive cells lacking Msx expression were also
observed (FIGS. 6A and 6B).
[0124] Based on PDX-1, Msx, and insulin staining patterns, at least
four populations of ductal cells can be identified in the
IFN.gamma. transgenic pancreas: PDX-1.sup.+ Msx.sup.- insulin.sup.-
ductal cells; PDX-1.sup.+ Msx.sup.+ insulin.sup.- ductal cells;
PDX-1.sup.+ Msx.sup.+ insulin.sup.+ ductal cells; and PDX-1.sup.+
Msx.sup.- insulin.sup.+ ductal cells.
[0125] In this study, histological analyses were used to
characterize the progenitor cells responsible for the ductal
proliferation and islet regeneration. Defined markers associated
with ducts during regeneration in the IFNg transgenic mouse
include: PDX-1 and Msx-2. These results suggest that these proteins
are associated with endocrine progenitor cells in the ducts of the
IFN.gamma. transgenic mouse.
[0126] The ductal epithelium has been designated as the site of
exocrine and endocrine development in the pancreas. A specific
pathway for islet development is thought to involve the derivation
of endocrine cells from duct cells, which progress through a series
of intermediate cell types. In promoting regeneration and new islet
formation, it is believed that progenitor stem cells in the ducts
of the IFN.gamma. transgenic mouse recapitulate the early
development of the pancreas. Parallels exist between normal
ontogeny and IFN.gamma.-mediated regeneration, and in both cases,
endocrine gene expression is an early event. Individual endocrine
cells are initially scattered in the duct wall; these cells
subsequently migrate to form clusters, which develop into fully
differentiated islets. Thus it was hypothesized that endocrine cell
precursors would be abundant in the ducts of mice undergoing islet
regeneration, as they are in the fetal pancreas.
[0127] Msx-2 is part of a conserved family of homeobox-containing
transcription factors that regulate tissue growth and patterning
during embryogenesis. Using the Atlas cDNA array to study
differential gene expression patterns, we found that Msx-2 is
expressed in the regenerating pancreas of the IFN.gamma. transgenic
mouse. We used immunohistochemistry to confirm these results and to
follow expression of the Msx protein directly. These experiments
utilized an antibody which detects both Msx-1 and Msx-2, preventing
us from drawing conclusions regarding Msx-2 expression specifically
from this staining alone. Despite this limitation, we detected
significant expression of Msx in the pancreatic ducts of IFN.gamma.
transgenic mice. This is in contrast to the work of others as well
as our own observations in non-transgenic mice demonstrating that
the Msx proteins are not expressed in the normal adult mouse
pancreas.
[0128] Strikingly, we also observed significant expression of Msx
in the developing pancreas during embryogenesis. This expression
was localized to the growing epithelia from which ducts and
endocrine cells arise. Coupled with the fact that we did not
observe enhanced Msx-1 expression in the transgenic pancreas using
the Atlas cDNA array, these results support our identification of
Msx-2 as a marker associated with endocrine progenitor cells both
in the developing and regenerating pancreas. Furthermore, our
staining demonstrated considerable cytoplasmic as well as nuclear
localization of Msx. As a transcription factor, Msx is expected to
be localized in the nucleus, although others have reported its
presence as a diffuse cytoplasmic stain as well.
[0129] A number of studies indicate that Msx-2 can be induced
through a signaling network involving members of the TGF beta
superfamily, including BMP4. TGF.alpha., IL1.beta., and TNF.alpha.
are elevated in the regenerating pancreas. Msx-2 is expressed at
many sites during development, including the cranial neural crest,
neural tube, tooth germs, eyes, ears, nose, limb buds, pituitary,
and heart, and, in particular, Msx-2 expression appears to be
associated with sites of epithelial-mesenchymal interactions. Msx
proteins are also thought to be crucial to pattern formation during
the development of diverse organs. A number of reports have also
implicated Msx-2 in the apoptotic program during development.
Although the expression of Msx-2 in the pancreas during development
has not been reported previously, the evidence presented here
suggests that Msx-2 plays a critical role in regulating the
pancreatic developmental program as well.
[0130] The identification of markers associated with endocrine
progenitor cells in the IFN.gamma. transgenic pancreas is clearly
of value, with regards to both defining these precursor cells and
in an analysis of the regenerative process. In this study we have
correlated the expression of two such molecules, PDX-1 and Msx-2,
with the striking pancreatic regeneration exhibited by the
IFN.gamma. transgenic mouse. Each of these homeodomain proteins
appears to play a critical role in organ formation during ontogeny,
and each is expressed in the developing as well as the regenerating
pancreas. While future studies will be aimed at defining the
precise contributions of these proteins during pancreatic
development and regeneration, their association with pancreatic
progenitor cells will be valuable in the isolation and
characterization of this critical cell type.
Example 3
[0131] Engraftment and Differentiation of In Vitro Cultured
Pancreatic Ductal Cells.
[0132] Whole pancreata were harvested from IFN.gamma. transgenic
and non-transgenic mice. Ductal fragments were subsequently
isolated and dispersed into cell suspensions. These ductal cell
preparations were cultured for four months. A large portion (50%)
of these cultured cells was found to express the ErbB3 receptor,
while a separate population of smaller cells was found to be
fibronectin positive, ErbB3 receptor negative. No endocrine cells
were present in these long-term cultures. Cultured cells were then
transplanted under the kidney capsule of recipient mice; each mouse
received two hundred thousand cells. The transplanted cells were
permitted to grow in vivo for three weeks, at which point the
recipient mice were sacrificed. Isolated kidneys were prepared for
immunohistochemical analysis. Staining for insulin expression
revealed the presence of insulin-producing cells within the grafts
(as shown in FIGS. 7A and 7B). These data indicate that cells
purified and cultured from pancreatic ducts and engrafted into
recipient mice can give rise to insulin-expressing cells at the
graft site. Similar results were obtained with experiments to
detect the presence of progenitors giving rise to glucagon
producing alpha cells.
[0133] The purification of ErbB3-expressing cells from the cultured
pancreatic duct preparations and their subsequent engraftment into
recipient mice may significantly enhance the extent of
insulin-producing cells generated within the graft.
[0134] All publications mentioned herein are incorporated herein by
reference for the purpose of describing and disclosing, for
example, the compounds and methodologies that are described in the
publications which might be used in connection with the presently
described invention. The publications discussed above and
throughout the text are provided solely for their disclosure prior
to the filing date of the present application. Nothing herein is to
be construed as an admission that the inventors are not entitled to
antedate such disclosure by virtue of prior invention.
* * * * *