Genetically altered human pluripotent stem cells

• US 20020168766A1
• Filed: 05/04/2001
• Published: 11/14/2002
• Est. Priority Date: 01/11/2000
• Status: Abandoned Application

• Current Assignee: Asterias Biotherapeutics Incorporated (Lineage Cell Therapeutics, Inc.)
• Original Assignee: Asterias Biotherapeutics Incorporated (Lineage Cell Therapeutics, Inc.)
• Inventors: Gold, Joseph D., Inokuma, Margaret S., Xu, Chunhui, Carpenter, Melissa K.
• Application Number:
  US09/849,022
• Publication Number:
  US 20020168766A1
• Time in Patent Office: Days
• US Class Current: 435/366
• CPC Class Codes: C12N 2501/01 Modulators of cAMP or cGMP, e.g. non-hydrolysable analogs, phosphodiesterase inhibitors, cholera toxin
• 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 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
• CPC Class Codes: C12Q 1/6881 for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
• CPC Class Codes: C12Q 2600/158 Expression markers

Abstract

This disclosure provides a system for obtaining genetically altered primate pluripotent stem (pPS) cells. The pPS cells are maintained in an undifferentiated state by culturing on a feeder cell line that has been immortalized and altered with drug resistance genes. Alternatively, 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 method of obtaining genetically altered primate pluripotent stem (pPS) cells or progeny thereof, comprising:

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

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

2. The method of claim 1, further comprising preferentially selecting cells that have been genetically altered with the polynucleotide.

3. The method of claim 1, 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.

5. The method of claim 1, 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.

6. The method of claim 1, 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.

7. The method of claim 1, wherein the pPS cells are human embryonic stem (hES) cells.

4. A method of producing genetically altered primate pluripotent stem (pPS) cells or progeny thereof, comprising:

a) providing a composition of pPS cells on a layer of feeder cells that are drug-resistant;

b) transferring a polynucleotide into pPS cells in the composition; and

c) selecting genetically altered cells in the composition using the drug to which the feeder cells are resistant.

8. An undifferentiated human pluripotent stem (hPS) cell genetically altered with a polynucleotide.

12. The composition of claim 8, wherein the pPS cells are human embryonic stem (hES) cells.

9. A stably transfected undifferentiated human pluripotent stem cell.

10. A population of primate pluripotent stem (pPS) cells, in which at least 25% of the undifferentiated pPS cells have been transfected with a polynucleotide, or are the progeny of such cells that have inherited the polynucleotide.

11. A population of genetically altered differentiated cells, obtained by differentiating the cells of claim 10.