Immortalized fibroblasts

• US 20070098696A1
• Filed: 10/31/2005
• Published: 05/03/2007
• Est. Priority Date: 10/31/2005
• Status: Active Grant

• Current Assignee: Centro De Investigacion Y De Estudios Avanzados Del I.P.N., President and Fellows of Harvard College (Harvard Corporation)
• Original Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Inventors: Green, Howard, Iuchi, Shiro, Marsch-Moreno, Meytha, Kuri-Harcuch, Walid
• Granted Patent: US 7,413,900 B2
• Time in Patent Office: Days
• US Class Current: 424/93.7
• CPC Class Codes: A61K 35/48 Reproductive organs
• CPC Class Codes: C12N 2502/02 embryonic cells
• CPC Class Codes: C12N 2502/13 connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
• CPC Class Codes: C12N 2510/04 Immortalised cells
• CPC Class Codes: C12N 5/0606 Pluripotent embryonic cells, e.g. embryonic stem cells [ES] embryonic germ cells C12N5/0611, induced pluripotent stem cells C12N5/0696
• CPC Class Codes: C12N 5/0652 Cells of skeletal and connective tissues; Mesenchyme

Abstract

Aspects of the invention provide an immortalized fibroblast cell line capable of the growth and maintenance of human embryonic stem cells. An immortalized fibroblast cell line derived from an early stage mouse embryo is provided. Methods of preparing an immortalized fibroblast cell line from an early stage mouse embryo are also provided.

Claims

1. A composition comprising

an immortalized fibroblast cell line established from a mouse embryo less than 15 days old and immortalized by serial passages.

2. The composition of claim 1, wherein the mouse embryo is between 12 and 13 days old.

3. The composition of claim 1, wherein the cell line, in a confluent layer, supports the growth and maintenance of Oct4 positive H9 human embryonic stem cells.

4. The composition of claim 1, wherein the cell line, in a confluent layer, supports the growth and maintenance of angular Oct4 positive H9 human embryonic stem cells, substantially free of dome-shaped cells.

5. The composition of claim 1, wherein the cell line contains a recombinant drug resistance gene.

6. The composition of claim 1, wherein the cell line is MMM cells, or progeny thereof, deposited with the ATCC under accession number ______.

7. A method for preparing an immortalized fibroblast cell line comprising

providing cells from a mouse embryo less than 15 days old;

and

passaging the cells serially to produce an immortalized fibroblast cell line.

8. The method of claim 7, wherein the mouse embryo is between 12 and 13 days old.

9. The method of claim 7, wherein the cells from the mouse embryo are passaged at least 20 times.

10. The method of claim 9, wherein the cells are passaged at least 30 times.

11. The method of claim 10, wherein the cells are passaged at least 40 times.

12. The method of claim 11, wherein the cells are passaged at least 50 times.

13. The method of claim 12, wherein the cells are passaged at least 60 times.

14. The method of claim 13, wherein the cells are passaged at least 65 times.

15. The method of claim 7, further comprising introducing a recombinant drug resistance gene into the immortalized cell line.

16. The method of claim 7, further comprising supporting the growth and maintenance of Oct4 positive H9 human embryonic stem cells that are substantially-free of dome-shaped cells, by seeding the H9 human embryonic stem cells on a confluent layer of immortalized fibroblast cells.

17. A composition comprising

an immortalized fibroblast cell line that when in a confluent layer, supports the growth and maintenance of Oct4 positive H9 human embryonic stem cells that are substantially free of dome-shaped cells.

18. The composition of claim 17, wherein the immortalized fibroblast cell line contains a recombinant drug resistance gene.

19. The composition of claim 17, wherein the immortalized fibroblast cell line supports the H9 human embryonic stem cells such that the majority of the stem cells are angular-shaped.

20. The composition of claim 17, wherein the immortalized fibroblast cell line is MMM cells, or progeny thereof, deposited with the ATCC under accession number ______.