Culturing pancreatic stem cells having a specified, intermediate stage of development

• US 6,759,039 B2
• Filed: 06/29/2001
• Issued: 07/06/2004
• Est. Priority Date: 06/30/2000
• Status: Expired due to Term

• Current Assignee: ReNeuron Limited (ReNeuron Group Plc)
• Original Assignee: Amcyte Inc. (ReNeuron Group Plc)
• Inventors: Tsang, Wen-Ghih, Zheng, Tianli, Huang, Chang Jiang
• Primary Examiner(s): Falk, Anne-Marie
• Assistant Examiner(s): Sullivan, Daniel M.
• Application Number:
  US09/895,585
• Publication Number:
  US 20020081725A1
• Time in Patent Office: 1,103 Days
• Field of Search: 424/93.7, 435/347, 435/435, 435/373, 435/375, 435/402
• US Class Current: 424/93.7
• CPC Class Codes: C12N 2501/305 Growth hormone [GH], aka. somatotropin
• CPC Class Codes: C12N 5/0676 Pancreatic cells
• CPC Class Codes: C12N 5/0677 Three-dimensional culture, tissue culture or organ culture; Encapsulated cells

Abstract

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 driven to late stage development, i.e. prototype islet cells. This invention further provides for culturing techniques that select for these intermediate differentiated stage cells and selectively eliminates early or late stage pancreatic cells.

Claims

1. A method of producing an aggregate of cultured pancreatic cells that comprises an encapsulating mantle of ck-19 positive cells and an inner cell mass, wherein the aggregate comprises 50-5000 pancreatic cells and has a diameter of between 50 and 300 microns, the method comprising the steps of

(a) culturing pancreatic cells on a substrate;

(b) removing the cells from the substrate;

(c) reseeding PDX-1 positive pancreatic cells on a substrate produced by step (b); and

(d) culturing the cells of step (c) on the substrate of step (b) to provide an aggregate of cultured pancreatic cells that comprise a surrounding mantle of ck-19 positive cells and an inner cell mass, wherein the aggregate comprises 50-5000 pancreatic cells and has a diameter of between 50 and 300 microns.

2. The method of claim 1, wherein the culturing of steps (a) or (c) takes place in a medium containing growth hormone and less than 1% total volume of serum.

5. An aggregate of pancreatic cells produced by the method of claim 1, wherein said aggregate secretes insulin.

7. A method of claim 1 wherein the cultured pancreatic cells are human cells.

8. An aggregate of claim 5 wherein the pancreatic cells are human cells.

3. A method of providing pancreatic endocrine function to a mammal, the method comprising the steps of

(a) producing an aggregate of cultured pancreatic cells by the method of step 7;

and

(b) implanting the aggregate within the mammal,

wherein implanting provides pancreatic endocrine function to the mammal.

4. A method of claim 3 where the cells are passaged in media containing between 1-2 mg per liter of recombinant growth hormone.

6. An aggregate of cultured pancreatic cells, comprising an encapsulating mantle of CK19-positive cells and an inner cell mass, wherein the aggregate comprises 50-5000 pancreatic cells, has a diameter of between 50 and 300 microns and secrete insulin.

9. An aggregate of claim 6 wherein the pancreatic cells are human cells.