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

• US 20020081725A1
• Filed: 06/29/2001
• Published: 06/27/2002
• Est. Priority Date: 06/30/2000
• Status: Active Grant

• Current Assignee: ReNeuron Limited (ReNeuron Group Plc)
• Original Assignee: Amcyte Inc. (ReNeuron Group Plc)
• Inventors: Tsang, Wen-Ghih, Zheng, Tianli, Huang, Chang Jiang
• Granted Patent: US 6,759,039 B2
• Time in Patent Office: Days
• US Class Current: 435/366
• 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 preparing a cell culture of propagating pancreatic cells having the ability to be passed from one culture vessel to a second vessel at an initial concentration of about 180 cells per square centimeter and expanded to about 1,800 cells per square centimeter, while retaining the following properties:

90% PDX-1 positive and having an insulin;

actin mRNA ratio of between 1;

100 and 1000;

1, said method comprising the steps of;

(a) isolating propagating pancreatic cells;

(b) transferring the cells to a culture medium containing growth hormone and having 1% or less total volume of serum to selectively propagate cells having an insulin;

actin mRNA ratio of between 1;

100 and 1000;

1 and that are PDX-1 positive; and

(c) passaging the cells to provide a cell culture of propagating pancreatic cells having the ability to be passed from one culture vessel to a second vessel at an initial concentration of about 180 cells per square centimeter and expanded to about 1,800 cells per square centimeter while retaining the following properties;

90% PDX-1 positive and having an insulin;

actin mRNA ratio of between 1;

100 and 1000;

1.

2. The method of claim 1, wherein the cells are maintained in a first medium containing serum at between 1% and 4% of the total volume of the first medium before transfer to a second medium containing growth hormone and 1% or less total volume of serum.

3. The method of claim 1, wherein the cells are maintained in a first medium containing serum at above 4% of the total volume of the first medium before transfer to a second medium containing growth hormone and 1% or less total volume of serum.

4. The method of claim 3, wherein the cells are transferred from a first medium containing serum at above 4% of the total volume of the serum to a second medium containing growth hormone and less than 1% of total volume of serum by successive transfers to media having successively lower amounts of serum.

5. The method of claim 1, wherein the isolated propagating pancreatic cells have a mixture of PDX-1 positive and PDX-1 negative phenotypes.

6. The method of claim 1, wherein the cell culture has an insulin:

actin mRNA ratio of between 1;

10 and 100;

1.

11. A method of claim 1 where the cells are passaged in media containing recombinant human growth hormone.

12. A method of claim 1 where the cells are passaged in media containing epithelial growth factor.

13. A culture of propagating pancreatic cells produced by the method of claim 1.

7. 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 (c) 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.

8. The method of claim 7, wherein the culturing of steps (a) or (c) takes place in a medium containing growth hormone and less than 1% total volume of serum.

15. An aggregate of pancreatic cells produced by the method of claim 7.

9. 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.

10. A method of claim 9 where the cells are passage in media containing between 1-2 mg per liter of recombinant growth hormone.

14. A culture of propagating pancreatic cells having the ability to be passed from one culture vessel to a second vessel at an initial concentration of about 180 cells per square centimeter and expanded to about 1,800 cells per square centimeter while retaining the following properties:

90% PDX-1 positive and having an insulin;

actin mRNA ratio of between 1;

100 and 1000;

1.

16. 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 and has a diameter of between 50 and 300 microns.