Selective antibody targeting of undifferentiated stem cells

• US 6,921,665 B2
• Filed: 11/26/2001
• Issued: 07/26/2005
• Est. Priority Date: 11/27/2000
• Status: Expired due to Fees

• Current Assignee: Asterias Biotherapeutics Incorporated (Lineage Cell Therapeutics, Inc.), Roslin Institute
• Original Assignee: Geron Corporation, Roslin Institute
• Inventors: Gold, Joseph D., McWhir, Jim, Schiff, J. Michael
• Primary Examiner(s): WOITACH, JOSEPH T
• Application Number:
  US09/995,419
• Publication Number:
  US 20030032187A1
• Time in Patent Office: 1,338 Days
• Field of Search: 435/72.1, 435/2, 435/7.1, 435/7.2, 435/6, 435/536, 435/23.2, 435/320.1, 424/95, 424/520, 424/590, 800/DIG.2, 514/44
• US Class Current: 435/455
• 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: A61K 48/00 Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• 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 2799/022 where the vector is derived from an adenovirus
• 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 9/1048 Glycosyltransferases (2.4)
• CPC Class Codes: C12N 9/1276 RNA-directed DNA polymerase (2.7.7.49), i.e. reverse transcriptase or telomerase
• CPC Class Codes: C12Y 204/01087 N-Acetyllactosaminide 3-alpha-galactosyltransferase (2.4.1.87), i.e. alpha-1,3-galactosyltransferase

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 expression of a cell-surface antigen that can be used to deplete the undifferentiated cells. Model effector sequences encode glycosyl transferases that synthesize carbohydrate xenoantigen or alloantigen, which can be used for immunoseparation or as a target for complement-mediated lysis. The differentiated cell populations produced are suitable for use in tissue regeneration and non-therapeutic applications such as drug screening.

Claims

1. A method of producing differentiated cells, comprising

a) obtaining a cell population comprising undifferentiated stem cells that have been genetically altered to contain a nucleic acid molecule comprising P-X, wherein X is nucleic acid sequence that causes expression of a cell surface antigen not normally expressed in the population, and P is a transcriptional control element operatively linked to X, such that the surface antigen is expressed in undifferentiated cells;

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

c) culturing the remaining differentiated cells.

2. The method of claim 1, wherein the undifferentiated stem cells are primate pluripotent stem (pPS) calls.

3. The method of claim 1, wherein X encodes a glycosyltransferase.

4. The method of claim 3, wherein X encodes an α

(1.3)galactosyltransferase.

5. The method of claim 3, wherein X encodes an A or B transferase from the ABO Blood Group system.

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

7. The method of claim 1, wherein the cells have been genetically altered using a vector comprising P-X.

8. The method of claim 1, wherein the cells have been genetically altered to place X Under control of a promoter (P) present in the cell genome.

9. The method of claim 1, further comprising depleting undifferentiated cells from the population by combining the cells the with a ligand specific for the antigen.

10. The method of claim 9, wherein the ligand is an antibody or a lectin.

11. The method of claim 9, comprising combining the cells with ligand specific for the antigen, and separating cells that have not bound the ligand.

12. The method of claim 9, comprising combining the cell population or progeny thereof with complement and antibody specific for the antigen under conditions that permit the complement to lyse cells to which the antibody has bound.

13. The method at claim 9, wherein a) comprise genetically altering the cell population such that P-X is transiently expressed in undifferentiated cells in the population.

14. The method of claim 13, wherein a) comprises genetically altering the cell population with an adenovirus vector comprising P-X.

15. The method of claim 9, wherein a) comprises genetically altering the cell population such that P-X is inherited by progeny of cells in the population, and thereby expressed in undifferentiated progeny.

16. The method of claim 15, wherein a) comprises genetically altering the cell population with a DNA plasmid or retrovirus vector comprising P-X.

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

a) obtaining a cell population that comprises both differentiated cells and undifferentiated stem cells;

b) genetically altering undifferentiated stem cells in the population so that they contain a nucleic acid molecule comprising P-X, wherein X is nucleic acid sequence that causes expression of a cell surface antigen not normally expressed in the population, and P is a transcriptional control element operatively linked to X, such that the surface antigen is expressed in the undifferentiated stem cells;

c) depleting undifferentiated cells from the population by combining the cells with a ligand specific for the antigen; and

d) culturing the remaining differentiated cells.

18. The method of claim 17, wherein the ligand is an antibody or a lectin.

19. The method of claim 17, comprising combining the cells with ligand specific for the antigen, and separating cells that have not bound the ligand.

20. The method of claim 17, comprising combining the cell population or progeny thereof with complement and antibody specific for the antigen under condition that permit the complement to lyse cells to which the antibody has bound.

21. The method of claim 17, wherein X encodes a glycosyltransferase.

22. The method of claim 17, wherein P is a TERT promoter.

23. The method of claim 17, wherein the promoter is an OCT-4 promoter.

24. A method for preparing cells, comprising:

a) obtaining human embryonic stem (hES) cells that have been genetically altered so as to transcribe a nucleic acid sequence under control of a promoter that preferentially drives transcription in undifferentiated hES calls, wherein transcription of the nucleic acid causes expression of a surface antigen not normally expressed by undifferentiated hES cell;

b) differentiating the hES cells; and

then

c) formulating the cells from step b) for administration to a mammalian host.

25. The method of claim 24, wherein the hES cells have been genetically altered to place the nucleic acid sequence under control of a promoter present in the cell genome.

26. The method of claim 24, wherein the hES cells have been genetically altered to place the nucleic acid sequence under control of a heterologous promoter.

27. The method of claim 24, wherein the promoter is a TERT promoter.

28. The method of claim 24, wherein the promoter is an OCT-4 promoter.

29. The method of claim 24, wherein the nucleic acid sequence encodes a cell surface protein not normally expressed in human cells.

30. The method of claim 24, wherein the nucleic acid encodes a glycosyltransferase that causes expression of a cell surface carbohydrate to which some humans have naturally occurring antibody.

31. The method of claim 24, wherein b) comprises differentiating the hES cells into neural cells.

32. The method of claim 24, wherein b) comprises differentiating the hES cells into hepatocytes.

33. The method of claim 24, further comprising depleting undifferentiated hES cells before the differentiated cells are formulated for administration to a mammalian host.

34. The method of claim 33, wherein the depleting step comprises adding a ligand specific for the antigen, and separating cells that have not bound the ligand.

35. The method of claim 33, wherein the depleting step comprises adding complement and antibody specific for the antigen under conditions that permit the complement to lyse cells to which the antibody has bound.

36. A method of depicting undifferentiated stem cells from a mixed cell population, comprising:

a) obtaining a mixed cell population that comprises both differentiated cells and undifferentiated human embryonic stem (hES) calls;

b) genetically altering the hES cells in the mixed cell population so as to transcribe a nucleic acid sequence under control of a promoter that preferentially drives transcription in undifferentiated hES cells, wherein transcription of the nucleic acid causes expression of a surface antigen not normally expressed by undifferentiated hES cells, thereby producing a genetically altered cell population;

c) depleting undifferentiated cells from the genetically altered cell population by using a lectin or antibody specific for the antigen, thereby producing a more homogeneous cell population; and

d) formulating the more homogeneous cell population for administration to a mammalian host.

37. The method of claim 36, wherein the promoter is a TERT promoter.

38. The method of claim 36, wherein the promoter is an OCT-4 promoter.

39. The method of claim 36, wherein c) comprises combining the genetically altered cell population with a lectin or antibody specific for the antigen, and separating cells that have not bound the lectin or antibody.

40. The method of claim 36, wherein c) comprises combining the genetically altered cell population with complement and antibody specific for the antigen under conditions that permit the complement to lyse cells to which the antibody has bound.