Morula derived embryonic stem cells

• US 20040229350A1
• Filed: 05/12/2003
• Published: 11/18/2004
• Est. Priority Date: 05/12/2003
• Status: Abandoned Application

• Current Assignee: Reproductive Genetics Institute Incorporated
• Original Assignee: Reproductive Genetics Institute Incorporated
• Inventors: Verlinsky, Yury, Strelchenko, Nikolai
• Application Number:
  US10/436,306
• Publication Number:
  US 20040229350A1
• Time in Patent Office: Days
• US Class Current: 435/366
• CPC Class Codes: C12N 2502/13 connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
• 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

A method for producing a human pluripotent embryonic stem cell line derived from culturing morula stage human embryo cells is disclosed. The method includes culturing the cells in close contact with a feeder cell layer to inhibit differentiation of the cells. A preparation of human pluripotent embryonic stem cells derived from culturing morula stage human embryo cells is also disclosed.

Claims

1. A method for producing a human pluripotent embryonic stem cell line comprising the steps of:

providing a morula stage human embryo cell;

positioning the morula cells onto a feeder cell layer;

culturing the morula cells to create multiple layers of cells;

passaging the multiple layers of cells onto a second culturing medium for the proliferation of embryonic stem cells.

2. The method of claim 1, wherein the morula cells include blastomeres.

3. The method of claim 1, wherein the morula cells are derived from enucleated oocytes after somatic cell nuclear transfer.

4. The method of claim 1, wherein the step of positioning the morula cells includes positioning in close contact with the feeder cell layer.

5. The method of claim 1, wherein the step of positioning the morula cells includes positioning underneath the feeder cell layer.

6. The method of claim 1, wherein the step ofpositioning the morula cells includes positioning between a plurality of feeder cell layers.

7. The method of claim 4, wherein the step of positioning the morula cells in close contact with the feeder cell layer prevents differentiation.

8. The method of claim 1, wherein the feeder cell layer is a mitotically inactive feeder layer.

9. The method of claim 1, wherein the step of passaging further includes isolation of individual cells.

10. The method of claim 1, wherein the step of passaging, further includes isolation of a cluster of cells.

11. The method of claim 9, wherein passaging of cells is accomplished by mechanical means.

12. The method of claim 1, wherein step of passaging the cells onto the second culturing medium prevents differentiation of the cells.

13. The method of claim 10, wherein the isolated cells are passaged onto another feeder cell layer.

14. The method of claim 12 wherein the isolated cells can be passaged indefinitely in an undifferentiated state onto a new culture medium creating an unlimited supply of ES cells.

15. The method of claim 1, further comprising the step of selecting embryonic stem cells with relatively low cytoplasm to nucleus ratios.

16. An embryonic stem cell line derived from the method of claim 1.

17. A method for producing a human pluripotent embryonic stem cell line comprising the steps of:

providing a morula stage human embryo cell;

removing a zona pellucida from a morula stage human embryo cell releasing a plurality of blastomeres;

positioning the blastomeres in close contact with a feeder cell layer;

culturing the blastomeres to create multiple layers of cells; and

passaging the multiple layers of cells onto a second culturing medium, wherein the second culturing medium enables further proliferation of cells and prevents differentiation of the resulting cells.

18. The method of claim 17, wherein the step of positioning the blastomeres includes positioning the blastomeres underneath the feeder cell layer.

19. The method of claim 17, wherein the step of positioning the blastomeres includes positioning the blastomeres between a plurality of feeder cell layers.

20. The method of claim 17, wherein the isolated cells are passaged onto another feeder cell layer.

21. An embryonic stem cell line derived from the method of claim 17.