Drug – bio-affecting and body treating compositions – Whole live micro-organism – cell – or virus containing – Animal or plant cell
Reexamination Certificate
2001-06-29
2004-07-06
Falk, Anne-Marie (Department: 1636)
Drug, bio-affecting and body treating compositions
Whole live micro-organism, cell, or virus containing
Animal or plant cell
C435S347000, C435S373000, C435S375000, C435S402000
Reexamination Certificate
active
06759039
ABSTRACT:
STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT
Not Applicable
REFERENCE TO A “SEQUENCE LISTING,” A TABLE, OR A COMPUTER PROGRAM LISTING APPENDIX SUBMITTED ON A COMPACT DISK.
Not Applicable
FIELD OF THE INVENTION
This invention relates to the discovery that an intermediate, differentiated stage of pancreatic stem cells exist that can be propagated in a stable manner in successive serial passaging while maintaining insulin production in response to glucose. These cells are advantageous in that they are both expandable and stable in culture and can be driven to late stage development. This invention further provides for culturing techniques that select for these intermediate differentiated stage cells and selectively eliminates early or late stage pancreatic cells.
BACKGROUND OF THE INVENTION
The mammalian pancreas develops from the embryonic foregut bud. As the embryonic buds grow, a ductal system develops by branching morphogenesis. After the ventral and dorsal anlage fuse, the new organ grows and matures into two interlocked structures, the exocrine system and the endocrine system. The majority of the pancreas is composed of acinar cells that produce digestive enzymes. The endocrine system includes &bgr;-cells, which produce insulin, &agr;-cells, which produce glucagon, and &dgr;-cells, which produce somatostatin. The endocrine cells are organized into clusters called islets.
Animal research has shown at least two mechanisms of &bgr;-cell formation: neogenesis from ductal precursor cells and replication of mature &bgr;-cells. Replication of differentiated &bgr;-cells is maintained postnatally into adulthood. Replication of &bgr;-cells is accelerated by an increased demand for insulin, for example, as a result of high glucose infusion, partial pancreatectomy, and during gestation. Under these conditions, &bgr;-cells mass quickly increases through both cell hypertrophy (enlargement of volume of individual cells) and hyperplasia (increase in the number of &bgr;-cells).
In Type I or insulin dependent diabetes mellitus (IDDM) there is a clear reduction in the number of &bgr;-cells due to an autoimmune attack against the &bgr;-cells. Eisenbarth,
N. Eng. J. Med.
314:1360-1368 (1986). A treatment for Type I diabetes could include increasing in the number of &bgr;-cells in a subject suffering from Type I diabetes. Bonner-Weir,
Endocrin.
141:1926-1929 (2000).
Another treatment for diabetes using islet cells involves grafting pancreatic tissue from immune matched donors into transplant recipients. Typically, transplant recipients are required to receive immunosuppressant therapy to prevent rejection of the transplanted organ. Recently developed immunosuppressant regimens have improved the results of clinical islet transplantation in humans. While the technique remains experimental, if islet cell transplants can perform the same function as whole organ pancreas grafts, this much simpler surgical procedure would play an important role in the treatment of diabetes.
Although the transplantation of human islets shows promise as a powerful treatment for diabetes, a number of impediments exist that presently limit the utility of this procedure. One significant impediment is the inability to produce sufficient numbers of islet cells for use in the procedure. Presently, the process used to obtain islets for transplantation typically involves isolation of pancreatic tissue, enzymatic digestion of the pancreatic tissue to liberate the individual cells from the surrounding tissue, and the use of a gradient centrifugation purification technique. The gradient centrifugation purification technique is well known in the art and is performed by many islet transplant centers. Unfortunately, the yield of islets from a single pancreas treated with the standard procedure is usually insufficient for transplantation. Accordingly, alternatives to this procedure have been sought and developed. The use of fetal tissue or xenogenic transplant tissue has been explored, but ethical issues, availability of source material, and concerns over immune rejection or xenotropic pathogenesis complicate such approaches.
To date the ability to isolate, culture, and expand pancreatic cells for use in transplantation to treat pancreatic endocrine disease has remained elusive. Although islet and islet cells can be isolated from pancreatic tissue, this isolated material remains viable and capable of endocrine function for only a short period of time if it is not properly preserved. Various approaches to isolating pancreatic stem cells and inducing differentiation in vitro have been reported (see Peck et al.,
Ann Med
33:186-192 (2001); Bonner-Weir et al.,
Proc Natl Acad Sci USA
14:7999-8004 (2000); U.S. Pat. Nos. 6,001,647; 5,928,942; 5,888,916; PCT publications WO 00/78929 and WO 00/47721). Previous methodologies, however, have suffered from several limitations. Expansion of the pancreatic cell population following isolation has generally required a period of growth in serum-containing medium (see. e.g., U.S. Pat. No. 5,888,916), which raises cost and safety issues. Moreover, while satisfactory cell proliferation is achieved by such methods, the resulting cell populations may not retain markers of pancreatic cell differentiation or the ability to produce insulin, and often cannot be consistently differentiated into viable and high hormone-producing cells.
Serum-free selective media, which can promote the growth of epithelial cell populations over less desirable cell types (see Stephan et al.,
Endocrinology
140:5841-54 (1999)), offer the possibility of overcoming some of these limitations. Serum-free culture conditions have been reported for culture of pancreatic cells isolated from adult tissues. See Bonner-Weir et al., supra; WO 00/78929. However, these procedures are not completely satisfactory. A period of culture in serum-containing medium, requiring special culture substrates, is still obligatory, and transition of the cells to a serum-free medium for differentiation eliminates their ability to propagate. What is required is a general method to isolate and culture pancreatic cellular material that consistently yields cells capable of proliferation in vitro while retaining the potential to produce pancreatic hormones. Such cell populations could reverse the diabetic state following transplantation, as well as serve as a source for pancreatic endocrine hormones in vitro, and provide model systems for the study of pancreatic development and disease. The present invention fulfills these and other needs.
SUMMARY OF THE INVENTION
This invention provides methods and compositions for culturing pancreatic cells in vitro. In one aspect, the invention provides a method of preparing a cell culture of propagating pancreatic cells, the method comprising the steps of isolating propagating pancreatic cells, transferring the cells to an epithelial-selective culture medium containing growth hormone and less than 1% total volume of serum, and culturing the cells through at least one passage in culture. This method yields a cell population capable of being expanded from about 180 cells per square centimeter to about 1,800 cells per square centimeter, which is characterized by the following properties: at least 90% of the cells are positive for the transcription factor PDX-1/IPF-1, and the insulin:actin mRNA ratio of the population is between 1:100 and 1000:1. In certain embodiments of the invention, at least 95%, 98%, 99%, or 100% of the cells stain positive for PDX-1. In one embodiment of the invention, the cells are capable of being expanded from about 90 cells per square centimeter and expanded to about 36,000 cells per square centimeter. In other embodiments, the insulin:actin mRNA ratio is between 1:10 and 100:1. In some embodiments of the invention, the insulin:actin mRNA ratio is the unstimulated level of insulin mRNA.
In one embodiment of the invention, prior to transfer of the cells to a culture medium containing less than 1% serum, the cells are maintained in a medium containing s
Huang Chang Jiang
Tsang Wen-Ghih
Zheng Tianli
AmCyte, Inc.
Falk Anne-Marie
Sullivan Daniel M.
Townsend and Townsend / and Crew LLP
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