Methods for sorting undifferentiated cells and uses thereof

• US 20070292836A1
• Filed: 05/16/2007
• Published: 12/20/2007
• Est. Priority Date: 03/28/2003
• Status: Abandoned Application

• Current Assignee: National Institute of Agrobiological Sciences
• Original Assignee: National Institute of Agrobiological Sciences
• Inventors: Tokunaga, Tomoyuki, Furusawa, Tadashi, Honda, Chris, Ohkoshi, Katsuhiro
• Application Number:
  US11/804,050
• Publication Number:
  US 20070292836A1
• Time in Patent Office: Days
• US Class Current: 435/1.1
• CPC Class Codes: C12N 15/873 Techniques for producing new embryos, e.g. nuclear transfer, manipulation of totipotent cells or production of chimeric embryos
• 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: G01N 33/566 using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds

Abstract

A method of sorting undifferentiated cells is provided. Subpopulations of cells contained in a single line of ES cells can be sorted using cell-surface markers. The sorted undifferentiated cells have ability to produce chimera with high contribution and germ line transmission efficiently. These undifferentiated cells can be used as a valuable vehicle for the production of transgenic animals and also knockout animals.

Claims

1. A method of sorting undifferentiated cells, wherein said method comprises contacting undifferentiated cells with an antibody to a cell-surface antigen, and sorting the undifferentiated cells according to the presence or absence of the binding to the antibody.

2. The method of claim 1, wherein the cell-surface antigen is selected from the group consisting of PECAM-1, SSEA-1, SSEA-3, and SSEA-4.

3. The method of claim 1, wherein the undifferentiated cells are embryonic stem (ES) cells derived from mammals or embryonic germ (EG) cells derived from mammals.

4. The method of claim 1, wherein the undifferentiated cells are transgenic.

5. The method of claim 2, wherein undifferentiated cells that bind to an antibody to PECAM-1 are sorted out.

6. The method of claim 5, wherein undifferentiated cells that bind to both an antibody to PECAM-1 and an antibody to SSEA-1 are sorted out.

7. The method of claim 5, wherein undifferentiated cells that bind to an antibody to PECAM-1, an antibody to SSEA-3, and an antibody to SSEA-4 are sorted out.

8. An undifferentiated cell obtained by the method of claim 5.

12. A host embryo comprising the undifferentiated cell of claim 8.

13. A host embryo comprising the undifferentiated cell of claim 8, wherein the embryo is constructed by injection of the undifferentiated cell of claim 8 into a fertilized embryo, or aggregation of that undifferentiated cell with a fertilized embryo.

14. A host embryo comprising the undifferentiated cell of claim 8, wherein said embryo is constructed by injection or fusion of the undifferentiated cell of claim 8 to an enucleated unfertilized oocyte.

15. A differentiated tissue complex derived from the undifferentiated cell of claim 8.

16. A differentiated tissue complex derived from the undifferentiated cell of claim 8, wherein said complex is constructed by inducing differentiation by in vitro culture or in vivo transplantation of the undifferentiated cell of claim 8.

17. A surrogate mother comprising the embryo of claim 12.

18. A fetus, baby, or descendants obtained from the surrogate mother of claim 17.

9. An isolated undifferentiated cell binding to an antibody to PECAM-1.

10. An isolated undifferentiated cell binding to an antibody to PECAM-1 and an antibody to SSEA-1.

11. An isolated undifferentiated cell binding to an antibody to PECAM-1, an antibody to SSEA-3, and an antibody to SSEA-4.

19. A method of preparing a chimeric embryo comprising the steps of:

a) introducing a ES cell which expresses PECAM-1 and SSEA-1 into a host embryo to be harvested; and

b) selecting a chimeric embryo obtained in step (a).

20. The method of claim 19, wherein the ES cell in step (a) is introduced into a host embryo by injecting a ES cell which expresses PECAM-1 and SSEA-1 into the host embryo.

21. The method of claim 19, wherein the ES cell in step (a) is introduced into a host embryo by aggregating a host embryo which a zona pellucida has been removed with a ES cell which expresses PECAM-1 and SSEA-1.

22. The method of claim 19 wherein the ES cell and host embryo are from mouse.

23. A method of preparing a chimeric mouse comprising the steps of:

a) transferring the embryo prepared by the method of claim 22 into a surrogate mother to be harvested; and

b) selecting a chimeric fetus born of the surrogate mother obtained in step (a).

24. A method of isolating undifferentiated cells which are capable of differentiating into an epiblast of a blastocyst comprising:

a) sorting a population of undifferentiated cells according to the presence or absence of the cell surface expression of PECAM-1; and

b) collecting the undifferentiated cells that express PECAM-1 on their cell surface, wherein said undifferentiated cells so, isolated differentiate into an epiblast of a blastocyst at a higher rate than unsorted undifferentiated cells.

25. The method of claim 24, wherein the undifferentiated cells so isolated are from mouse and further express SSEA-1 on their cell surface.

26. The method of claim 24, wherein the undifferentiated cells are embryonic stem (ES) cells derived from mammals or embryonic germ (EG) cells derived from mammals.

27. The method of claim 24, wherein the undifferentiated cells so isolated are transgenic mouse undifferentiated cells.

28. The method of claim 24, wherein the presence or absence of the cell surface expression of PECAM-1 is detected by binding of an antibody to PECAM-1.

29. The method of claim 25, wherein the expression of SSEA-1 on the mouse undifferentiated cells so isolated is detected by binding of an antibody to SSEA-1.