Methods for providing differentiated stem cells

• US 6,576,464 B2
• Filed: 02/13/2001
• Issued: 06/10/2003
• Est. Priority Date: 11/27/2000
• Status: Expired due to Term

• Current Assignee: Asterias Biotherapeutics Incorporated (Lineage Cell Therapeutics, Inc.)
• Original Assignee: Geron Corporation
• Inventors: Lebkowski, Jane S., Gold, Joseph D.
• Primary Examiner(s): Crouch, Deborah
• Assistant Examiner(s): WOITACH, JOSEPH T
• Application Number:
  US09/783,203
• Publication Number:
  US 20020098582A1
• Time in Patent Office: 847 Days
• Field of Search: 435/284, 435/325, 536/23.1, 536/23.4, 536/25.5, 536/24.1
• US Class Current: 435/325
• 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 primate pluripotent stem (pPS) cell containing a nucleic acid molecule comprising the structure P-X, wherein:

X is a nucleic acid sequence 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

P is a transcriptional control element that causes X to be preferentially expressed in undifferentiated pPS cells.

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

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

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

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

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

8. The stem cell of claim 1, wherein the nucleic acid molecule contains the structure P-X-Y, wherein Y is a nucleic acid sequence that imparts drug resistance to a cell in which it is expressed, and wherein P causes both X and Y to be preferentially expressed in undifferentiated pPS cells.

9. The stem cell of claim 1, which is a human embryonic stem (hES) cell.

10. A method of producing a population of differentiated cells, comprising

a) obtaining a cell population comprising undifferentiated stem cells that contain 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 P is a transcriptional control element that causes X to be preferentially expressed in undifferentiated pPS cells;

and

b) causing at least some undifferentiated cells in the population to differentiate.

11. The method of claim 10, further comprising combining the cell population with the external agent.

12. A method of depleting a cell population of undifferentiated stem cells, comprising genetically altering undifferentiated stem cells in the population so that they contain 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, and P is a transcriptional control element that causes X to be preferentially expressed in undifferentiated cells.

13. The method of claim 10, wherein X encodes a toxin, or a protein that induces or mediates apoptosis.

14. A method of depleting a cell population of undifferentiated stem cells, comprising:

a) genetically altering undifferentiated stem cells in the population so that they contain a nucleic acid molecule comprising the structure P-X, wherein X renders a cell in which it is expressed susceptible to a lethal effect of an external agent, and P is a transcriptional control element that causes X to be preferentially expressed in undifferentiated cells; and

b) depleting undifferentiated cells from the population by contacting the cells with the external agent.

15. The method of claim 10, wherein X encodes an enzyme that converts a prodrug to a compound that is lethal to a cell in which X is expressed.

16. The method of claim 10, wherein X encodes a thymidine kinase.

17. The method of claim 16, wherein the external agent is ganciclovir.

18. The method of claim 10, wherein P-X is an introduced heterologous molecule.

19. The method of claim 10, wherein P is an endogenous transcriptional control element.

20. The method of claim 10, wherein the cell population is genetically altered such that X is transiently expressed in undifferentiated cells in the population.

21. The method of claim 10, wherein P-X is inherited by progeny of cells in the population, becoming expressed in undifferentiated progeny.

22. The method of claim 10, wherein P is an OCT-4 promoter or a promoter of telomerase reverse transcriptase (TERT).

23. The method of claim 10, wherein the stem cells are human embryonic stem (hES) cells.

24. The method of claim 10, wherein the nucleic acid molecule contains the structure P-X-Y, wherein Y is a nucleic acid sequence that imparts drug resistance to a cell in which it is expressed, and wherein P causes both X and Y to be preferentially expressed in undifferentiated pPS cells.

25. A plurality of cells according to claim 1, making up a cell population that contains less than 0.02% undifferentiated pPS cells.

26. The method of claim 14, comprising causing cells in the population to differentiate into neural cells or hepatocytes before contacting the cells with the external agent.

27. The stem cell of claim 1, wherein P causes X to be expressed at a level that is at least 5-fold higher in undifferentiated pPS cells.

28. The stem cell of claim 1, wherein P causes X to be expressed at a level that is at least 25-fold higher in undifferentiated pPS cells.

29. The method of claim 10, wherein P causes X to be expressed at a level that is at least 5-fold higher in undifferentiated pPS cells.

30. The method of claim 10, wherein P causes X to be expressed at a level that is at least 25-fold higher in undifferentiated pPS cells.