Human pancreatic epithelial progenitor cells and methods of isolation and use thereof

• US 6,436,704 B1
• Filed: 04/10/2000
• Issued: 08/20/2002
• Est. Priority Date: 04/10/2000
• Status: Expired due to Term

• Current Assignee: MacroGenics, Inc.
• Original Assignee: Raven Technology (Beijing) Co., Ltd.
• Inventors: Roberts, Penelope E., Mather, Jennie Powell
• Primary Examiner(s): Crouch, Deborah
• Assistant Examiner(s): Woitach, Joseph T.
• Application Number:
  US09/546,577
• Time in Patent Office: 862 Days
• Field of Search: 424/93.7, 424/93.1, 435/325, 435/378, 435/1.1, 435/363, 435/405, 435/383, 800/8, 514/44
• US Class Current: 435/366
• CPC Class Codes: A61K 2035/122 for inducing tolerance or supression of immune responses
• CPC Class Codes: A61K 2035/126 Immunoprotecting barriers, e.g. jackets, diffusion chambers
• CPC Class Codes: A61K 2039/515 Animal cells
• CPC Class Codes: A61K 35/12 Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells vaccines or medicinal preparations containing antigens or antibodies A61K39/00
• CPC Class Codes: A61P 1/18 for pancreatic disorders, e.g. pancreatic enzymes
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: C12N 2500/25 Insulin-transferrin; Insulin-transferrin-selenium
• CPC Class Codes: C12N 2500/90 Serum-free medium, which may still contain naturally-sourced components
• CPC Class Codes: C12N 2501/11 Epidermal growth factor [EGF]
• CPC Class Codes: C12N 2501/39 Steroid hormones
• CPC Class Codes: C12N 2501/392 Sexual steroids
• CPC Class Codes: C12N 2501/395 Thyroid hormones
• CPC Class Codes: C12N 2501/60 Transcription factors
• CPC Class Codes: C12N 5/0678 Stem cells; Progenitor cells; Precursor cells

Abstract

The invention discloses a substantially pure population of human pancreatic progenitor cells and methods of isolating and culturing the pancreatic progenitor cells. By carefully manipulating the microenvironment of the pancreatic progenitor cells, multiple passages are attainable wherein the pancreatic progenitor cells do not senesce and furthermore, are capable of becoming functional exocrine or endocrine cells. In addition, several methods of use of human pancreatic progenitor cells are disclosed herein.

Claims

1. A substantially pure population of human pancreatic progenitor cells, wherein said population of pancreatic progenitor cells will differentiate into acinar, ductal, and islet cells.

2. The population of pancreatic progenitor cells according to claim 1, wherein the pancreatic progenitor cells are isolated and maintained in serum-free media.

3. The population of pancreatic progenitor cells according to claim 1, wherein said pancreatic progenitor cells are identifiable by the expression of at least one cell marker.

4. The population of pancreatic progenitor cells according to claim 3, wherein said cell marker is selected from the group consisting of cytokeratin-19, carcinoembryonic antigen, carbonic anhydrase II, and cystic fibrosis transmembrane conductance regulator.

5. The population of pancreatic progenitor cells according to claim 4, wherein said pancreatic progenitor cells have the morphology of small and round, about 10 μ

m across the cell, and in a highly compacted columnar epithelial form.

6. The population of pancreatic progenitor cells according to claim 5, wherein said pancreatic progenitor cells can further differentiate into acinar cells expressing amylase and wherein said acinar cells have the appearance of large clusters forming acini.

7. The population of pancreatic progenitor cells according to claim 5 wherein said pancreatic progenitor cells can further differentiate into ductal cells expressing cytokeratin 19 and wherein said ductal cells have the morphology of small, round, about 40 μ

m across the cell, and a compacted, cuboidal columnar epithelial form.

8. The population of pancreatic progenitor cells according to claim 5 wherein said pancreatic progenitor cells can further differentiate into islet cells expressing insulin and glucagon and wherein said islet cells have the appearance of epithelial islands surrounded by acinar exocrine units.

9. A method of isolating the substantially pure population of human pancreatic progenitor cells of claim 1, comprising the steps of:

(a) microdissecting a source of human fetal pancreatic progenitor cells to yield a mixed population of pancreatic cells comprising pancreatic progenitor cells;

(b) placing the mixed population of pancreatic cells in nutrient media under culture conditions sufficient to sustain life of said pancreatic progenitor cells and wherein the nutrient media contains nutrients consisting of;

insulin, transferrin, epidermal growth factor, ethanolamine, phosphoethanolamine, selenium, triiodothyronine, progesterone, hydrocortisone, forskolin, heregulin, aprotinin, and bovine pituitary extract;

(c) maintaining suitable culture conditions sufficient to allow pancreatic progenitor cells to form aggregate or monolayer formation; and

(d) subculturing said aggregate or monolayer formation to effect isolation of the substantially pure population of pancreatic progenitor cells.

10. A method of providing a source of an immunogen to a heterologous recipient, comprising introducing a plurality of pancreatic progenitor cells as recited in claim 1 in an amount effective to induce an immune response in said recipient.

11. A method of generating human pancreatic tissue models in an immunodeficient or immunocompromised non-human mammalian recipient, comprising the step of administering human pancreatic progenitor cells of claim 1, which have been recombined ex vivo with mesenchymal tissue able to effect differentiation of said pancreatic progenitor cells, into said recipient at a location within said recipient able to support growth of said pancreatic progenitor cells.

12. A method of providing a source of pancreatic tissue-specific biological components in a pharmaceutical development of one or more drugs comprising isolating the population of human pancreatic progenitor cells as recited in claim 1, and using said pancreatic progenitor cells or any cellular part of the cells thereof as targets of one or more drugs under development.

13. A method of providing a source of nucleic acids or proteins in a development of bioassays comprising isolating nucleic acids or proteins from the human pancreatic progenitor cells as recited in claim 1 and using said nucleic acids or proteins as one or more of the principle component in the bioassays.