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 embryonic stem (ES) cells from a blastomere, comprising:
(a) providing an isolated mammalian blastomere;
(b) culturing the isolated blastomere in the presence of E-cadherin and
(i) pluripotent cells, and/or
(ii) fibronectin, tenascin, elastin, laminin, vitronectin, syndecan, or proteoglycan, or a combination thereof,
until said blastomere gives rise to a cluster of progeny cells; and
(c) transferring the cluster of progeny cells to ES cell medium and culturing the cluster of progeny cells therein to form a culture of mammalian ES cells from the cluster of progeny cells.
2. The method of claim 1 , wherein the mammalian blastomere is a human blastomere.
3. The method of claim 1 , wherein the blastomere is cultured in the presence of a fibronectin, a tenascin, an elastin, a laminin, a vitronectin, a syndecan, a proteoglycan, or a combination thereof.
4. The method of claim 1 , wherein the blastomere is cultured in the presence of pluripotent cells comprising embryonic stem cells, embryonic germ cells, or embryonic carcinoma cells.
5. The method of claim 1 , wherein the blastomere is cultured in step (b) in a medium containing less than 5 mM glucose.
6. The method of claim 1 , wherein the blastomere is cultured in step (b) in a medium containing less than 5 mM glucose and having an osmolarity less than 310 mOsm/kg.
7. The method of claim 1 , wherein the ES cell medium contains at least 5 mM glucose and has an osmolarity of at least 310 mOsm/kg.
8. The method of claim 1 , wherein the blastomere is cultured in the presence of a laminin.
9. An in vitro method of producing embryonic stem (ES) cells from a blastomere, comprising:
(a) providing an isolated mammalian blastomere;
(b) culturing the isolated blastomere in the presence of E-cadherin with or without pluripotent cells until said blastomere gives rise to a cluster of progeny cells; and
(c) transferring the cluster of progeny cells to ES cell medium and culturing the cluster of progeny cells therein to form a culture of mammalian ES cells from the cluster of progeny cells.
10. The method of claim 9 , wherein the blastomere is cultured in the presence of a laminin.
11. An in vitro method of producing embryonic stem (ES) cells from a blastomere, comprising:
(a) providing an isolated mammalian blastomere;
(b) culturing the isolated blastomere in a medium containing less than 5 mM glucose in the presence of
(i) pluripotent cells and/or
(ii) an extracellular matrix component comprising fibronectin, tenascin, elastin, laminin, vitronectin, syndecan, or proteoglycan, or a combination thereof,
until said blastomere gives rise to a cluster of progeny cells; and
(c) transferring the cluster of progeny cells to ES cell medium and culturing the cluster of progeny cells therein to form a culture of mammalian ES cells from the cluster of progeny cells.
12. An in vitro method of producing embryonic stem (ES) cells from a blastomere, comprising:
(a) providing an isolated mammalian blastomere;
(b) culturing the isolated blastomere in the presence of
(i) pluripotent cells and/or
(ii) an extracellular matrix component comprising fibronectin, tenascin, elastin, laminin, vitronectin, syndecan, or proteoglycan, or a combination thereof,
until said blastomere gives rise to a cluster of progeny cells; and
(c) transferring the cluster of progeny cells to ES cell medium and culturing the cluster of progeny cells therein to form a culture of mammalian ES cells from the cluster of progeny cells,
wherein the blastomere and/or the cluster of progeny cells are cultured in a medium containing less than 5 mM glucose and having an osmolarity of less than 310 mOsm/kg.