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. An in vitro method of producing human embryonic stem (ES) cells from a blastomere, comprising:
(a) providing a blastomere isolated from a human embryo;
(b) culturing the blastomere until said blastomere gives rise to a cluster of two or more progeny blastomeres;
(c) culturing the cluster of two or more progeny blastomeres with human pluripotent cells to obtain a culture of human ES cells that originate from the cluster of progeny blastomeres;
(d) isolating the human ES cells that originate from the cluster of progeny blastomeres of step (c).
2. The method of claim 1 , wherein the culture of step (b) comprises culturing said blastomere isolated from the human embryo of step (a) with the remaining human embryo from which said blastocyst was isolated.
3. The method of claim 1 , wherein step (b) comprises culturing in a medium containing less than 5 mM glucose.
4. The method of claim 1 , wherein step (b) comprises culturing in a medium containing less than 5 mM glucose and having an osmolarity less than 310 mOsm/kg.
5. The method of claim 1 , wherein step (b) comprises culturing in a medium having an osmolarity between about 260 and about 280 mOsm/kg.
6. The method of claim 1 , wherein step (b) comprises culturing in a medium containing less than 5 mM glucose and having an osmolarity between about 260 and about 280 mOsm/kg.
7. The method of claim 1 , wherein the cluster of two or more progeny blastomeres obtained in step (b) contains 16 or fewer blastomeres.
8. The method of claim 1 , wherein the human embryo of step (a) is between the two cell stage and the stage prior to compaction of the morula.
9. The method of claim 1 , wherein the human embryo of step (a) is between the four-cell stage and the sixteen-cell stage.
10. The method of claim 1 , wherein the human embryo of step (a) is undergoing compaction of the morula.
11. The method of claim 1 , wherein the pluripotent cells of step (c) are selected from the group consisting of ES cells, embryonic germ cells, and embryonic carcinoma cells.
12. The method of claim 1 , wherein step (c) comprises culturing in a medium selected from the group consisting of: media containing at least 5 mM glucose, media having an osmolarity of at least 310 mOsm/kg, and a medium containing at least 5 mM glucose and having an osmolarity of at least 310 mOsm/kg.
13. The method of claim 1 , wherein said providing step of (a) yields said blastomere and a viable human embryo from which the blastomere was obtained.
14. The method of claim 13 , wherein the remaining human embryo is cryopreserved following obtaining the blastomere.
15. The method of claim 1 , wherein the blastomere is obtained by immobilizing the embryo and tapping the immobilized embryo until the blastomere is isolated.
16. The method of claim 1 , further comprising performing genetic testing on at least one progeny cell of said blastomere.
17. An in vitro method of producing human embryonic stem (ES) cells from a blastomere, comprising:
(a) providing a blastomere isolated from a human embryo;
(b) culturing the blastomere in a culture comprising two or more human blastomeres until said blastomere isolated from the human embryo gives rise to a cluster of two or more progeny blastomeres; and
(c) culturing the cluster of two or more progeny blastomeres with human ES cells, thereby producing a culture containing of human ES cells that originate from the cluster of two or more progeny blastomeres;
(d) isolating the human ES cells that originate from the cluster of two or more progeny blastomeres.
18. The method of claim 17 , wherein step (b) comprises culturing in a medium having an osmolarity between about 260 and about 280 mOsm/kg.
19. The method of claim 17 , wherein the cluster of two or more progeny blastomeres obtained in step (b) contain 16 or fewer blastomeres.
20. The method of claim 17 , wherein the human embryo of step (a) is between the two-cell stage and the stage where the embryo is undergoing compaction of the morula.
21. The method of claim 17 , wherein step (c) comprises culturing in a medium selected from the group consisting of: media containing at least 5 mM glucose, media having an osmolarity of at least 310 mOsm/kg, and a medium containing at least 5 mM glucose and having an osmolarity of at least 310 mOsm/kg.
22. The method of claim 17 , wherein said providing step of (a) yields said blastomere and a viable human embryo from which the blastomere was obtained.
23. The method of claim 22 , wherein the remaining human embryo is cryopreserved following obtaining the blastomere.
24. The method of claim 17 , wherein the blastomere is obtained by immobilizing the embryo and tapping the immobilized embryo until the blastomere is isolated.
25. The method of claim 17 , further comprising performing genetic testing on at least one progeny cell of said blastomere.
26. The method of claim 17 , wherein step (b) comprises culturing in a medium containing less than 5 mM glucose and having an osmolarity less than 310 mOsm/k.