Multicellular living organisms and unmodified parts thereof and – Nonhuman animal – Transgenic nonhuman animal
Reexamination Certificate
1998-12-16
2001-06-05
Hauda, Karen M. (Department: 1632)
Multicellular living organisms and unmodified parts thereof and
Nonhuman animal
Transgenic nonhuman animal
C800S013000, C800S014000
Reexamination Certificate
active
06242666
ABSTRACT:
TECHNICAL FIELD
This invention relates to generally to models for identifying and isolating pancreatic stem cells, and in one embodiment, to models for identifying and isolating a common stem/progenitor cell that gives rise to hepatic cells and pancreatic cells.
BACKGROUND OF THE INVENTION
A healthy adult pancreas is usually a developmentally stable organ, but a targeted cell loss or aberrant cell growth and development have pathological consequences. For example, in human Insulin Dependent Diabetes Mellitus (IDDM), autoimmune mechanisms cause the selective and permanent destruction of the insulin-producing beta cells in the islets of Langerhans. The lost cells are not restored in vivo, although Sarvetnick et al.
Cell
52:773-782 (1988) have shown that beta cells have the potential to regenerate. On the other hand, the proliferation of duct cells can contribute to pancreatic disease pathologies, such as chronic pancreatitis, pancreatic cancer, and cystic fibrosis. Pancreatic duct cell proliferation and differentiation have also been shown by Arnush et al.,
Lab Invest
74: 985-990 (1995) to play a critical role in a transgenic mouse model of islet regeneration. Further, endocrine cell differentiation is frequently seen in pancreatic duct cell carcinomas, suggesting that duct cell proliferation can lead to islet neogenesis.
Growth factors are critical for modulating cellular proliferation and differentiation. The growth factors that regulate and promote pancreatic growth are not well characterized, but keratinocyte growth factor (KGF), a member of the fibroblast growth factor family, is known to be involved in wound healing and in the differentiation of many epithelial tissues. KGF upregulates epithelial cell proliferation and pancreatic duct cell proliferation in rats. Also, epidermal growth factor (EGF) and transforming growth factor beta-1 (TGF&bgr;-1) can induce ductal and endocrine cell development, respectively. EGF, which is known to stimulate epithelial cell and fibroblast proliferation, also has mitogenic properties for pancreatic growth. The overexpression of EGF and EGF receptor (EGF-R) is linked both to chronic pancreatitis and to malignant pancreatic growth.
Yi et al. (
Am J Pathol
145: 80-85, 1994) have shown that systemic administration of KGF to rats induces pancreatic duct cell proliferation. KGF stimulated the proliferation of pancreatic ductal epithelial cells in rats after daily systemic injection for 1-2 weeks. Duct cell proliferation was predominantly adjacent to or within the islets of Langerhans and occurred in the absence of physical injury to the pancreas. However, knockout mice lacking KGF do not display significant developmental abnormalities, and pancreatic and liver development appear entirely normal (Guo et al.,
EMBO J
12: 973-986, 1993).
Clearly, KGF can in some way influence pancreatic growth, and further studies designed to investigate its role in this process would be of considerable value. It is therefore important to expand our knowledge of these growth factors by providing animal models allowing the study of liver and pancreatic growth and development, and associated disease states.
SUMMARY OF THE INVENTION
The invention provides animal model systems for identifying pancreatic stem/progenitor cells, and in one embodiment, a common stem/progenitor cell which gives rise to liver cells (hepatocytes) and pancreatic cells. The animal model ectopically expresses KGF from a pancreatic specific promoter, for example, the insulin promoter (ins-KGF). Transgenic animals that express KGF in pancreatic islets of Langerhans have enlarged islets, with substantial proliferation of duct cells within the islet mass. The animals also have albumin and alpha-fetoprotein-producing hepatocytes in the islets. The transgenic animals show no pathology, hyperglycemia, or hypoglycemia associated with the KGF expression.
The invention further provides transgenic EGF and EGF×KGF animals, in both of which the ectopic expression of the growth factors is under the control of a pancreatic specific promoter. Animals with beta cell-targeted expression of EGF show significant morphological changes, including cellular proliferation and disorganized islet of Langerhans growth. EGF×KGF transgenic animals experience more profound changes in pancreatic morphology than do single transgenic animals. Double transgenic animals also show proliferation of pancreatic cells and extensive intra-islet fibrosis, which increases in severity with time. The animals of the invention are useful for the study of the effects of EGF and KGF on pancreatic growth and development, and for the study of pathologies associated with aberrant overexpression of EGF or KGF.
The invention provides several distinct cell types, including pancreatic hepatocytes, derived from a common stem/progenitor to liver cells and pancreatic cells. The invention also provides transgenic duct cells, and transgenic amylase-producing cells. The invention further provides methods of making and proliferating the transgenic cells and methods of using the transgenic cells.
The invention provides for the proliferation and differentiation of common stem/progenitor cells in vivo. A common stem/progenitor to liver cells and pancreatic cells is contacted in vivo with a developmentally effective amount of a growth factor. The growth factor induces the growth factor-responsive cells to differentiate. When the genetic modification is for the production of a biologically active substance, the substance will generally be one that is useful for the treatment of a given disorder. The invention thus provides methods for inducing specific cell types, including hepatocytes, duct cells, and amylase-producing cells. An interesting aspect of these methods of the invention is the underlying scientific information regarding the development of the specific cells in the pancreas. This useful scientific information results from the generation of the animal models of the invention.
The invention provides a method for the transfection of a common stem/progenitor to liver cells and pancreatic cells with vectors which can express the gene products for growth factors or growth factor receptors.
The invention provides a pancreactic duct culture. The pancreatic duct culture is useful for the development of a stem cell assay for identifying a common stem/progenitor cell in pancreatic islets.
The invention provides a method for using the homeobox protein PDX-1 as a marker for identifying a common stem/progenitor to liver cells and pancreatic cells. Antibodies to PDX-1 are used to revealed the presence of PDX-1 nuclear staining in a subset of ductal cells bordering the lumen, as well as in a subset of perilumenal ductal cells. No differences are observed in morphology between those ductal cells which expressed PDX-1 and those which did not, but insulin expression, characteristic of the newly formed islet structure, is present in a subpopulation of PDX-1 expressing ductal cells.
DETAILED DESCRIPTION
Introduction The invention provides animals, where the expression of EGF, KGF, or both EGF and KGF is under the control of a pancreatic specific promoter, e.g., the insulin promoter. As such, the invention provides an animal model system for identifying and isolating pancreatic stem/progenitor cells and for identifying and isolating a cell which is a common stem/progenitor for liver cells (hepatocytes) and pancreatic cells.
A definition of a “stem cell” is provided by Potten & Loeffler,
Development,
110:1001 (1990), who have defined stem cells as “undifferentiated cells capable of (a) proliferation, b) self-maintenance, (c) the production of a large number of differentiated functional progeny, (d) regenerating the tissue after injury, and (e) a flexibility in the use of these options.” Stem cells arc used in a body to replace cells that are lost by natural cell death, injury or disease. The presence of stem cells in a particular type of tissue usually correlates with tissues that have a high turnover of cells, stem cells arc also present in tissues, e.g
Krakowski Michelle L.
Kritzik Marcie R.
Sarvetnick Nora
Hauda Karen M.
Mintz Levin Cohn Ferris Glovsky and Popeo P.C.
The Scripps Research Institute
Woitach Joseph T.
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