Isolated in vitro cell population comprising primate pluripotent stem cells containing a nucleic acid construct and differentiated progeny of the pluripotent stem cells

• US 9,023,645 B2
• Filed: 03/15/2013
• Issued: 05/05/2015
• Est. Priority Date: 11/27/2000
• Status: Expired due to Fees

• Current Assignee: Asterias Biotherapeutics Incorporated (Lineage Cell Therapeutics, Inc.)
• Original Assignee: Asterias Biotherapeutics Incorporated (Lineage Cell Therapeutics, Inc.)
• Inventors: Gold, Joseph D, Lebkowski, Jane S
• Primary Examiner(s): Crouch, Deborah
• Application Number:
  US13/837,522
• Publication Number:
  US 20130273651A1
• Time in Patent Office: 781 Days
• US Class Current: 435/366
• CPC Class Codes: A61K 35/12 Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells vaccines or medicinal preparations containing antigens or antibodies A61K39/00
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C12N 15/85 for animal cells
• 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

This invention provides a system for producing differentiated cells from a stem cell population for use wherever a relatively homogenous cell population is desirable. The cells contain an effector gene under control of a transcriptional control element (such as the TERT promoter) that causes the gene to be expressed in relatively undifferentiated cells in the population. Expression of the effector gene results in depletion of undifferentiated cells, or expression of a marker that can be used to remove them later. Suitable effector sequences encode a toxin, a protein that induces apoptosis; a cell-surface antigen, or an enzyme (such as thymidine kinase) that converts a prodrug into a substance that is lethal to the cell. The differentiated cell populations produced according to this disclosure are suitable for use in tissue regeneration, and non-therapeutic applications such as drug screening.

Claims

1. An isolated in vitro cell population comprising differentiated cells and less than 1% primate pluripotent stem (pPS) cells, wherein the differentiated cells are the progeny of the pPS cells, and wherein the differentiated cells and the pPS cells comprise a nucleic acid molecule comprising the structure P-X,

wherein X is a nucleic acid sequence encoding a product that is lethal to a cell in which it is expressed or renders a cell in which it is expressed susceptible to a lethal effect of an external agent, and

wherein P is a transcriptional control element that is expressed in the pPS cells and down regulated when the pPS cells are induced to differentiate.

2. The cell population of claim 1, wherein X encodes a toxin, or a protein that induces or mediates apoptosis.

3. The cell population of claim 1, wherein X encodes an enzyme that converts a prodrug to a compound that is lethal to a cell in which X is expressed.

4. The cell of population claim 3, wherein X encodes a thymidine kinase.

5. The cell population of claim 1, wherein P-X is an introduced heterologous molecule.

6. The cell population of claim 1, wherein P is an endogenous transcriptional control element.

7. The cell population of claim 1, wherein P is an OCT 4 promoter or a promoter of telomerase reverse transcriptase (TERT).

8. The cell population of claim 1, wherein the differentiated cells are neurons.

9. The cell population of claim 1, wherein the differentiated cells are hepatocytes.

10. The cell population of claim 1, wherein the differentiated cells are cardiomyocytes.

11. The cell population of claim 1, wherein the cell population comprises less than 0.2% pPS cells.

12. The cell population of claim 1, wherein the cell population comprises less than 0.05% pPS cells.

13. The cell population of claim 1, wherein the cell population comprises less than 0.01% pPS cells.

14. The cell population of claim 1, wherein the cell population comprises less than 20 ppm pPS cells.

15. The cell population of claim 1, wherein the cell population comprises less than 5 ppm pPS cells.

16. The cell population of claim 11, wherein the pPS cells are human embryonic stem cells.

17. The cell population of claim 12, wherein the pPS cells are human embryonic stem cells.

18. The cell population of claim 13, wherein the pPS cells are human embryonic stem cells.

19. The cell population of claim 14, wherein the pPS cells are human embryonic stem cells.

20. The cell population of claim 15, wherein the pPS cells are human embryonic stem cells.