Methods of making pancreatic islet cells

Details Methods of making pancreatic islet cells Methods of making pancreatic islet cells

2000-06-23

2004-11-09

Afremova, Vera (Department: 1651)

Chemistry: molecular biology and microbiology

Animal cell, per se ; composition thereof; process of...

Method of regulating cell metabolism or physiology

C435S375000, C435S384000, C435S366000, C435S370000

Reexamination Certificate

active

06815203

ABSTRACT:

BACKGROUND OF THE INVENTION
The term “idiopathic diabetes mellitus” covers a heterologous group of disorders having common symptomatic characteristics. These symptoms include an absolute or relative insulin deficiency, fasting hyperglycemia, glycosuria and a tendency to develop arteriosclerosis, neuropathy and nephropathy. At least two major as well as several less common variants of the disease have been identified. One of the major types of diabetes is insulin-dependent diabetes mellitus (IDDM) or Type 1 diabetes which covers about 10% of patients having diabetes. The other major type of diabetes, non-insulin dependent diabetes mellitus (NIDDM) or Type 2 diabetes, represents the remaining 90% of patients having diabetes.
Absent regular insulin replacement therapy using exogenously produced insulin and/or careful monitoring of diet, diabetes patients experience a wide range of debilitating symptoms, which can progress to coma and ultimately death.
An alternative method of treating diabetes which does not require repeated administration of insulin and/or strict monitoring of diet is the transplantation of pancreatic cells or tissue from a donor to a diabetic patient. However, a major problem with pancreatic cell tissue transplantation is the shortage of human donor tissue. Only about 3,000 cadaver pancreases become available in the United States each year while about 35,000 new cases of Type 1 diabetes are diagnosed each year. Hering et al. (1999) Graft 2:12-27.
SUMMARY OF THE INVENTION
The present invention is based, in part, on the discovery that pancreatic duct and exocrine cells are capable of serving as precursor cells. It was found that by replication, mature duct and/or exocrine cells can revert to a less differentiated cell that can then redifferentiate into islet, exocrine or mature duct cells and that external signals direct the phenotypic differentiation of these cells. Thus, pancreatic duct cells can provide a source of islet cells which can be used in transplantation procedures.
Accordingly, in one aspect, the invention features a method of promoting dedifferentiation of pancreatic cells. The method includes: obtaining a population of adult or differentiated pancreatic cells; and allowing the adult or differentiated cells to proliferate, e.g., rapidly proliferate, e.g., proliferate in the presence of an agent which promotes expansion., thereby providing dedifferentiated pancreatic cells.
In a preferred embodiment, the population of adult or differentiated pancreatic cells can be: a population substantially free of islet cells, e.g., a population from which the islet cells have been removed or have been substantially removed. In a preferred embodiment, the pancreatic cells are human pancreatic cells. In a preferred embodiment, the population of cells includes: duct cells; exocrine cells; duct and exocrine cells; less than about 60%, 50%, 40%, 30%, 20%, 10%, 5%, 1% islet cells. In a preferred embodiment, the population of cells is obtained form cells remaining after islet isolation.
In a preferred embodiment, the population of cells is selected based on the ability to attach to a container, e.g., a culture flask, e.g., a non-sticky culture flask. These cells are also referred to herein as “adherent cells”. In another preferred embodiment, the cells that do not attach to the container are removed from the container and cultured in another container until until the cells attach. Preferably, the cells that do not attach to the container are removed when at least 1%, 2%, 3%, 5%, 10%, 15%, 20% or more of the surface of the container has cells attached to it. Once the cells attach, they can be used in the methods of the invention. In a preferred embodiment, the adherent cells express low levels or no insulin, e.g., the cells express less than about 600 ng, 500 ng, 400 ng, 300 ng, 200 ng, 150 ng, 100 ng, 50 ng of insulin. In a preferred embodiment, the adherent cells have: less than about 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 3%, 2%, 1% the insulin content of an original sample of cells obtained from a pancreas or pancreases; less than about 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 3%, 2%, 1% the DNA content of an original sample of cells obtained from a pancreas or pancreases.
In a preferred embodiment, the agent which promotes expansion is: a polypeptide or fragment or analog thereof which binds TGF-&bgr;, e.g., a soluble TGF-&bgr; receptor; an antibody which binds TGF-&bgr;; an nucleic acid which binds to TGF-&bgr; and inhibits TGF-&bgr; expression, e.g., a TGF-&bgr; antisense molecule; at least one growth factor; combinations thereof.
In a preferred embodiment, the method includes providing an agent which promotes proliferation of adult or differentiated pancreatic cells. Preferably, the agent is a growth factor or a combination of growth factors. The growth factor can be one or more of: keratinocyte growth factor (KGF); epidermal growth factor (EGF); transforming growth factors (TGF-&agr;); hepatocyte growth factor (HGF). Preferably, the growth factor is KGF.
In a preferred embodiment, the cells are allowed to proliferate by placing the cells on a substrate, e.g., a container, e.g., a plastic container, with medium containing an agent which promotes proliferation of adult or differentiated pancreatic cells, e.g., a growth factor, e.g., KGF, EGF, TGF-&agr;, and/or HGF. In a preferred embodiment, the growth factor is a growth factor which promotes the proliferation of pancreatic duct cells, e.g., rapid proliferation of pancreatic duct cells.
In a preferred embodiment, the container is: a plastic container, e.g., a plastic flask, e.g., a non-sticky plastic flask; a plastic container wherein an extracellular matrix protein has been laid down in the container, e.g., plastic container, e.g., plastic flask. In a preferred embodiment, the extracellular matrix protein is laid down by a cell, e.g., the extracellular matrix is laid down by a cancer derived cell line, e.g., a bladder carcinoma cell line, e.g., an A431 cell line. In another preferred embodiment, the extracellular matrix protein is: added to the container; is a laminin, e.g., laminin 5; is a collagen, e.g., collagen I and/or collagen IV.
In a preferred embodiment, the cells are placed on a substrate in a glucose-containing media, e.g., the glucose-containing media comprises about 4 mM, 6 mM, 8 mM, 10 mM glucose. The media can be serum free. In a preferred embodiment, nicotinamide is added to the media; insulin/transferrin/selenium (ITS) is added to the media; bovine serum albumin (BSA) is added to the media; combinations of nicotinamide, ITS and/or BSA is added to the media.
In a preferred embodiment, the population of cells is: cultured until confluent; cultured until semi-confluent; cultured until the cells form a monolayer. In a preferred embodiment, the population of cells is cultured until at least about 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% confluency.
In a preferred embodiment, the population of cells is cultured for at least 1, 2, 3, 5, 10, 14, 18, 20, 25, 30 or more days.
In a preferred embodiment, the dedifferentiated pancreatic cells express a marker indicative of expansion. The marker can be one or more of: cytokeratin; PDX-1; IPF-1; Pref-1; lack of insulin.
In another aspect, the invention features a method of obtaining pancreatic islet cells from dedifferentiated pancreatic cells. The method includes adding an extracellular matrix component to a population of dedifferentiated pancreatic cells; and culturing the cells, to thereby obtaining pancreatic islet cells.
In a preferred embodiment, the population of dedifferentiated cells includes: dedifferentiated duct cells; dedifferentiated exocrine cells; both dedifferentiated duct cells and dedifferentiated exocrine cells. In a preferred embodiment, the cells are human cells.
In a preferred embodiment, the population of cells: is a monolayer of cells; has been cultured until semi-confluent; has been cultured until confluent. In a preferred embodiment, Lp; the population of cells has been cultured until at least about 40%, 50%, 60%

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