Derivation of embryonic stem cells and embryo-derived cells

• US 9,617,512 B2
• Filed: 04/30/2014
• Issued: 04/11/2017
• Est. Priority Date: 11/04/2004
• Status: Active Grant

• Current Assignee: Astellas Institute for Regenerative Medicine (Astellas Pharma Incorporated)
• Original Assignee: Astellas Institute for Regenerative Medicine (Astellas Pharma Incorporated)
• Inventors: Chung, Young Gie, Lanza, Robert P., Klimanskaya, Irina V.
• Primary Examiner(s): Noble, Marcia S
• Application Number:
  US14/266,114
• Publication Number:
  US 20140356952A1
• Time in Patent Office: 1,077 Days
• CPC Class Codes: A61K 35/12 Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells vaccines or medicinal preparations containing antigens or antibodies A61K39/00
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C12N 15/873 Techniques for producing new embryos, e.g. nuclear transfer, manipulation of totipotent cells or production of chimeric embryos
• CPC Class Codes: C12N 2500/34 Sugars
• CPC Class Codes: C12N 2501/115 Basic fibroblast growth factor (bFGF, FGF-2)
• CPC Class Codes: C12N 2501/998 Proteins not provided for elsewhere
• CPC Class Codes: C12N 2502/02 embryonic cells
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 5/0603 Embryonic cells production of embryos, nuclear transfer A01K67/027; Embryoid bodies
• CPC Class Codes: C12N 5/0604 Whole embryos; Culture medium therefor
• 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

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.

Claims

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.