Embryonic stem cells having genetic modifications

• US 20050164385A1
• Filed: 09/24/2004
• Published: 07/28/2005
• Est. Priority Date: 10/23/1998
• Status: Active Grant

• Current Assignee: Asterias Biotherapeutics Incorporated (Lineage Cell Therapeutics, Inc.)
• Original Assignee: Geron Corporation
• Inventors: Gold, Joseph D., Inokuma, Margaret S., Xu, Chunhui, Carpenter, Melissa K.
• Granted Patent: US 7,413,904 B2
• Time in Patent Office: Days
• US Class Current: 435/368
• CPC Class Codes: C12N 2501/13 Nerve growth factor [NGF]; Brain-derived neurotrophic factor [BDNF]; Cilliary neurotrophic factor [CNTF]; Glial-derived neurotrophic factor [GDNF]; Neurotrophins [NT]; Neuregulins
• CPC Class Codes: C12N 2501/155 Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
• CPC Class Codes: C12N 2502/13 connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
• CPC Class Codes: C12N 2502/99 genetically modified cells
• CPC Class Codes: C12N 2503/02 Drug screening
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 2510/00 Genetically modified cells
• 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 5/0619 Neurons
• CPC Class Codes: C12N 5/0622 Glial cells, e.g. astrocytes, oligodendrocytes; Schwann cells

Abstract

This disclosure provides a system for obtaining genetically altered primate pluripotent stem (pPS) cells. The role of the feeder cells is replaced by supporting the culture on an extracellular matrix, and culturing the cells in a conditioned medium. The cells can be genetically altered with a viral vector or DNA/lipid complex, and then selected for successful transfection by drug-resistant phenotype in the transfected cells. The system allows for bulk proliferation of genetically altered pPS cells as important products for use in human therapy or drug screening.

Claims

1. A population of substantially undifferentiated genetically modified human embryonic stem cells.

2. The cell population of claim 1, growing in a culture environment that is essentially free of feeder cells.

3. The cell population of claim 1, wherein the cells have been modified so as to express luciferase, green fluorescent protein, alkaline phosphatase, β

-galactosidase, neomycin, puromycin, neophospho-transferase, or human telomerase reverse transcriptase.

4. The cell population of claim 1, wherein the cells have been modified with so as to express a gene under control of an EF1a promoter or a PGK promoter.

5. The cell population of claim 1, wherein the genetic modification is stable.

6. The cell population of claim 1, wherein over 25% of the cells in the cell population have the genetic alteration.

7. The cell population of claim 1, wherein over 75% of the cells in the cell population have the genetic alteration.

8. A method of obtaining genetically modified human embryonic stem (hES) cells according to claim 1, comprising:

a) providing a culture of pPS cells essentially free of feeder cells; and

b) transferring a polynucleotide into pPS cells in the culture.

9. The method of claim 8, further comprising preferentially selecting cells that have been genetically altered with the polynucleotide.

10. The method of claim 8, wherein the hPS cells are cultured in an environment comprising extracellular matrix components and a conditioned medium produced by collecting medium from a culture of feeder cells.

11. The method of claim 8, wherein the polynucleotide comprises a protein encoding region operably linked to a promoter that promotes transcription of the encoding region in an undifferentiated pPS cell.

12. The method of claim 8, wherein the polynucleotide is selected from the group consisting of an adenoviral vector, a retroviral vector, and a DNA plasmid complexed with positively charged lipid.

13. A method of obtaining a population of genetically modified differentiated cells, comprising differentiating the stem cells of claim 1.

14. The method of claim 8, wherein the differentiated cells are neural cells.

15. The method of claim 8, wherein the differentiated cells are hepatocytes.

16. A method of obtaining genetically modified differentiated cells, comprising differentiating the genetically modified human embryonic stem cells of claim 1.

17. The method of claim 16, wherein the cells are differentiated into hepatocytes.

18. The method of claim 16, wherein the cells are diferentiated into neurons.

19. A method of drug screening, comprising contacting the cells of claim 1 with a test compound, and determining what effect the test compound has on the cells

20. A method of drug screening, comprising differentiating the stem cells of claim 1, contacting the differentiated cells with a test compound, and determining what effect the test compound has on the differentiated cells.