Feeder-free culture method for embryonic stem cell

• US 20030175956A1
• Filed: 12/24/2002
• Published: 09/18/2003
• Est. Priority Date: 10/23/1997
• Status: Active Grant

• Current Assignee: Asterias Biotherapeutics Incorporated (Lineage Cell Therapeutics, Inc.)
• Original Assignee: Andrea G. Bodnar, Choy-Pik Chiu, James T. Murai, Joseph D. Gold, Margaret Inokuma, Michael D. West
• Inventors: West, Michael D., Gold, Joseph D., Murai, James T., Bodnar, Andrea G., Chiu, Choy-Pik, Inokuma, Margaret
• Granted Patent: US 7,413,902 B2
• Time in Patent Office: Days
• US Class Current: 435/366
• CPC Class Codes: C12N 2500/40 Nucleotides, nucleosides, bases cyclic nucleotides C12N2501/01, anti-neoplasic drugs C12N2501/06
• CPC Class Codes: C12N 2500/44 Thiols, e.g. mercaptoethanol
• CPC Class Codes: C12N 2501/385 of the family of the retinoic acid recptor, e.g. RAR, RXR; Peroxisome proliferator-activated receptor [PPAR]
• CPC Class Codes: C12N 2502/13 connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
• CPC Class Codes: C12N 2510/00 Genetically modified 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

Abstract

Methods and materials for culturing primate-derived primordial stem cells are described. In one embodiment, a cell culture medium for growing primate-derived primordial stem cells in a substantially undifferentiated state is provided which includes a low osmotic pressure, low endotoxin basic medium that is effective to support the growth of primate-derived primordial stem cells. The basic medium is combined with a nutrient serum effective to support the growth of primate-derived primordial stem cells and a substrate selected from feeder cells and an extracellular matrix component derived from feeder cells. The medium further includes non-essential amino acids, an anti-oxidant, and a first growth factor selected from nucleosides and a pyruvate salt.

Claims

1. A method of causing primate primordial stem (pPS) cells to proliferate in an undifferentiated form, comprising culturing them on an extracellular matrix with a nutrient medium in a growth environment free of feeder cells.

2. The method of claim 1, wherein the extracellular matrix is a fibroblast matrix, prepared by culturing fibroblasts, lysing the fibroblasts in situ, and then washing what remains after lysis.

3. The method of claim 1, wherein the extracellular matrix is a matrix prepared from a matrix component or combination of such components.

4. The method of claim 1, wherein the extracellular matrix is prepared from an isolated matrix component or a combination of components selected from collagen, placental matrix, fibronectin, laminin, merosin, tenascin, heparin sulfate, condroitin sulfate, dermatan sulfate, aggrecan, biglycan, thrombospondin, vitronectin, and decorin.

5. The method of claim 1, wherein the nutrient medium comprises sodium pyruvate and nucleosides, and has a low endotoxin level.

6. The method of claim 1, wherein the nutrient medium comprises one or more exogenously added factors that promote undifferentiated growth of primordial stem cells, selected from TGF-β

, IL-11, IL-6, IL-6 receptor, IL-1, LIF, IL-17, LAP, MCP-1, bFGF, FGF-4, PDGF soluble receptor A, forskolin, and antibodies to IL-8, TGF-β

, BDNF, TNF-β

, VEGF, and EGF.

7. The method of claim 1, wherein the nutrient medium contains added fibroblast growth factor.

8. The method of claim 1, wherein the nutrient medium contains added forskolin.

9. The method of claim 1, wherein the nutrient medium is a conditioned medium.

10. The method of claim 9, wherein the conditioned medium is obtained by collecting medium from a culture of growing feeder cells.

11. The method of claim 1, comprising passaging the pPS cells into a new growth environment such that the pPS cells are distributed on a solid surface in a manner that permits them to proliferate without differentiating.

12. The method of claim 11, wherein the pPS cells are distributed such that the cells are in clusters.

13. The method of claim 11, wherein the new growth environment comprises extracellular matrix components but no feeder cells.

14. The method of claim 1, further comprising testing the pPS cells for alkaline phosphatase activity.

15. The method of claim 1, further comprising testing the pPS cells for expression of SSEA-1 or SSEA-4.

16. The method of claim 1, further comprising testing the pPS cells for expression of telomerase.

17. The method of claim 1, further comprising adding an additional component to the culture environment, and ascertaining whether the component helps maintain the pPS cells in an undifferentiated form.

18. The method of claim 17, wherein the component is a matrix component.

19. The method of claim 17, wherein the component is a soluble factor.

20. The method of claim 17, further comprising causing or permitting the pPS cells to differentiate.