Derivation of embryonic stem cells and embryo-derived cells
View Patent ↗This present invention provides novel methods for deriving embryonic stem cells and embryo-derived cells from an embryo without requiring destruction of the embryo. The invention further provides cells and cell lines derived without embryo destruction, and the use of the cells for therapeutic and research purposes. It also relates to novel methods of establishing and storing an autologous stem cell line prior to implantation of an embryo, e.g., in conjunction with reproductive therapies such as IVF.
1. A method of producing human embryonic stem (ES) cells, comprising:
(a) culturing at least one human blastomere to generate a cluster of two or more progeny blastomeres;
(b) culturing the cluster of two or more progeny blastomeres with human pluripotent cells or in a medium conditioned with human pluripotent cells;
(c) culturing the cluster of two or more blastomeres of (b) to produce a culture containing human ES cells that originate from the at least one human blastomere; and
(d) isolating the human ES cells that originate from the at least one human blastomere.
2. The method of claim 1 , wherein step (c) comprises culturing the cluster of two or more progeny blastomeres in a medium containing at least 5 mM glucose and having an osmolarity of at least 310 mOsm/kg.
3. The method of claim 1 , wherein the at least one blastomere in step (a) comprises two or more blastomeres.
4. The method of claim 1 , wherein the at least one blastomere in step (a) comprises two or more blastomeres obtained from the same human embryo.
5. The method of claim 1 , wherein the at least one blastomere in step (a) comprises two or more blastomeres obtained from different human embryos.
6. The method of claim 1 , wherein the at least one blastomere in step (a) is obtained from a human embryo which is not destroyed after said at least one blastomere is obtained.
7. The method of claim 6 , wherein the at least one blastomere in step (a) is obtained from a human embryo which is viable after said at least one blastomere is obtained.
8. The method of claim 1 , wherein the at least one human blastomere in step (a) is obtained from a human embryo before or during compaction of the morula.
9. The method of claim 1 , wherein the at least one blastomere in step (a) is obtained from a 4-10 cell embryo.
10. The method of claim 1 , wherein step (b) comprises culturing the cluster of two or more progeny blastomeres in indirect contacted with human pluripotent cells.
11. The method of claim 1 , where recombinant Oct-4 is introduced into the blastomeres or endogenous Oct-4 is activated in the blastomeres during step (c).
12. The method of claim 1 , wherein step (a) comprises culturing said at least one human blastomere in a medium containing less than 5 mM glucose and having an osmolarity of less than 310 Osm/kg.
13. The method of claim 1 , wherein step (b) comprises culturing the cluster of two or more progeny blastomeres with a medium conditioned with human pluripotent cells.
14. The method of claim 1 , wherein the medium in step (b) comprises adrenocorticotropic hormone (“ACTH”).
15. The method of claim 1 , wherein the cluster of two or more progeny blastomeres obtained in step (a) contains 16 or fewer blastomeres.
16. The method of claim 1 , wherein step (b) comprises culturing the cluster of two or more progeny blastomeres in a medium conditioned with human pluripotent cells selected from the group consisting of human ES cells, embryonic germ cells, and embryonic carcinoma cells.