Composite blastocysts (CBs) from aggregates of dissociated cells of non-viable pre-embryos

• US 20030087431A1
• Filed: 11/08/2001
• Published: 05/08/2003
• Est. Priority Date: 11/08/2001
• Status: Active Grant

• Current Assignee: Mina Alikani, Steen Malte Willadsen
• Original Assignee: Mina Alikani, Steen Malte Willadsen
• Inventors: Willadsen, Steen Malte, Alikani, Mina
• Granted Patent: US 6,759,244 B2
• Time in Patent Office: Days
• US Class Current: 435/366
• 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 2503/04 Screening or testing on artificial tissues
• 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

A preparation and a method of making composite blastocysts (CBs) from aggregates of dissociated cells of non-viable pre-embryos are disclosed. The CB is characterized morphologically by having two distinct tissue types, the inner cell mass (ICM) and the trophectoderm (TE), and a blastocoelic cavity (BC). The ICM is differentially stainable with bisbenzimide and the TE is differentially stainable with propidium iodide. The ICM is pluripotent in that it contains embryonic stem (ES) cells. The TE cells are pluripotent in that they can give rise to all cell types normally derived from TE cells. The primate TE is characterized by the production of chorionic gonadotrophin. The method of making CBs is an aggregation process (AP) comprising inter alia the following steps: 1) dissociation of discarded pre-embryos; 2) isolation of single nucleated cells from dissociated discarded pre-embryos; 3) microsurgical encapsulation of several cells within a host zona pellucida or artificial aggregation with or without a non-zona vessel; and 5) primary culture of the cell aggregates for multiplication and differentiation of cells.

One particularly advantageous embodiment is that the starting material is non-viable pre-embryos. Another advantageous embodiment is that the AP allows individual cells from non-viable pre-embryos to further multiply, and become integrated into CBs. The novel CBs and the novel aggregation process are disclosed.

Claims

1. A preparation of composite blastocysts (CBs) comprising cells derived from non-viable pre-embryos.

2. A preparation of CBs of claim 1 which maintain the potential to differentiate.

3. A preparation of CBs of claim 1 wherein the CB maintains the potential to differentiate into derivatives of ectoderm, mesoderm, and endoderm tissues.

4. The preparation of CBs of claim 1 comprising cells derived from non-viable primate pre-embryos.

5. The preparation of CBs of claim 1 comprising cells derived from non-viable human pre-embryos.

6. The preparation of CBs of claim 1 comprising cells derived from non-viable and viable human pre-embryos.

7. A stem cell line isolated from the preparation of claim 1.

8. A stem cell line isolated from the preparation of claim 6.

13. A method of isolating stem cell lines comprising the method of claim 1.

9. A method of producing CBs comprising the steps of

a) dissociation of non-viable pre-embryos into non-nucleated and individual nucleated cells or groups of cells;

b) isolation of individual mononucleated cells or groups of mononucleated cells from disaggregated non-viable pre-embryos;

c) aggregation of isolated mononucleated cells or groups of mononucleated cells from non-viable pre-embryos in a host zona pellucida;

d) culturing of the zona-encapsulated cell aggregates to allow multiplication and differentiation of cells.

10. The method of claim 9 wherein aggregation of the nucleated cells does not involve the use of a host zona pellucida.

11. The method of claim 9 wherein one or more of the nucleated cells are derived from a viable embryo.

12. A method of isolating stem cell lines comprising the method of claim 9.