Oxygen tension for the parthenogenic activation of human oocytes for the production of human embryonic stem cells
View Patent ↗Methods of producing human stem cells are disclosed for parthenogenetically activating human oocytes by manipulation of O 2 tension, including manipulation of Ca 2+ under high O 2 tension and contacting oocytes with serine threonine kinase inhibitors under low O 2 tension, isolating inner cell masses (ICMs) from the activated oocytes, and culturing the cells of the isolated ICMs under high O 2 tension. Moreover, methods are described for the production of stems cells from activated oocytes in the absence of non-human animal products, including the use of human feeder cells/products for culturing ICM/stem cells. Stem cells produced by the disclosed methods are also described.
1. A method of producing human stem cells comprising:
a) parthenogenetically activating a human oocyte, wherein activating comprises: i) contacting the oocyte with an ionophore at high O 2 tension and ii) contacting the oocyte with a serine-threonine kinase inhibitor under low O 2 tension;
b) cultivating the activated oocyte of step (a) at low O 2 tension until blastocyst formation;
c) transferring the blastocyst to a layer of feeder cells, and culturing the transferred blastocyst under high O 2 tension;
d) mechanically isolating an inner cell mass (ICM) from trophectoderm of the blastocyst of step (c); and
e) culturing the cells of the ICM of step (d) on a layer of feeder cells, wherein culturing step (e) is carried out under high O 2 tension, thereby producing human stem cells.
2. The method of claim 1 , wherein low O 2 tension is maintained by incubation in a gas mixture environment comprising an O 2 concentration of about 2% O 2 to about 5% O 2 .
3. The method of claim 2 , wherein the gas mixture environment further comprises about 5% CO 2 and about 90% N 2 to 93% N 2 .
4. The method of claim 1 , wherein high O 2 tension is maintained by incubation in a gas mixture environment comprising about 5% CO 2 and about 20% O 2 .
5. The method of claim 1 , wherein the ionophore is selected from the group consisting of ionomycin and A23187.
6. The method of claim 1 , wherein the serine-threonine kinase inhibitor is selected from the group consisting of staurosporine, 2-aminopurine, sphingosine, and 6- dimethylaminopurine (DMAP).
7. The method of claim 1 , wherein the activating, isolating, and culturing steps are carried out under defined media conditions for therapeutic applications.
8. The method of claim 7 , wherein the media comprises human umbilical cord serum.
9. The method of claim 8 , wherein the media comprises about 10% human umbilical cord serum.
10. The method of claim 1 , wherein the layer of feeder cells comprises human fibroblasts.
11. The method of claim 10 , wherein the fibroblasts are postnatal human dermal fibroblasts.
12. The method of claim 10 , wherein the feeder cells are inactivated with an antibiotic.
13. The method of claim 12 , wherein the antibiotic is mitomycin C.
14. A method of activating a human metaphase II oocyte comprising;
a) incubating a human metaphase II oocyte in in vitro fertilization (IYF) media;
b) incubating the cell of step (a) in IVF media comprising an ionophore;
c) incubating the cell of step (b) in IYF media comprising a serine-threonine kinase inhibitor; and
d) incubating the cells of step (c) in fresh IVF medium until blastocyst formation,
wherein the incubating steps (a) and (b) are carried out under high O 2 tension, and wherein an inner cell mass (ICM) obtained from the blastocyst at step (d) produce culturable stem cells.
15. The method of claim 14 , wherein the O 2 tension for incubating steps (c) and (d) is maintained by incubating the cells in a gas mixture environment comprising an O 2 concentration of about 2% O 2 to 5% O 2 .
16. The method of claim 15 , wherein the gas mixture environment further comprises about 5% CO 2 and about 90% N 2 to 93% N 2 .
17. The method of claim 14 , further comprising incubating the oocytes with hyaluronidase.
18. The method of claim 14 , wherein incubating step (a) is carried out for about 2 hours at about 37° C.
19. The method of claim 14 , wherein incubating step (b) is carried out for about 5 minutes at about 37° C.
20. The method of claim 14 , wherein incubating step (c) is carried out for about 4 hours at about 37° C.
21. The method of claim 14 , wherein incubating step (d) is carried out for about 24 hours at about 37° C.
22. The method of claim 14 , wherein the IVF media is free of non-human products.
23. The method of claim 22 , wherein the ionophore is selected from the group consisting of ionomycin and A23187.
24. The method of claim 23 , wherein the ionophore is ionomycin.
25. The method of claim 22 , wherein the serine-threonine kinase inhibitor is selected from the group consisting of staurosporine, 2-aminopurine, sphingosine, and 6- dimethylaminopurine (DMAP).
26. The method of claim 25 , wherein the serine-threonine kinase inhibitor is DMAP.