Gynogenetic or androgenetic production of pluripotent cells and cell lines, and use thereof to produce differentiated cells and tissues

• US 9,249,388 B2
• Filed: 02/07/2013
• Issued: 02/02/2016
• Est. Priority Date: 10/28/1999
• Status: Expired due to Fees

• Current Assignee: University Of Massachusetts
• Original Assignee: University Of Massachusetts
• Inventors: Robl, James M., Cibelli, Jose, Burnside, Amy
• Primary Examiner(s): Noble, Marcia S
• Application Number:
  US13/761,897
• Publication Number:
  US 20130309206A1
• Time in Patent Office: 1,090 Days
• US Class Current: 1/1
• 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: A61K 35/545 Embryonic stem cells; Pluripotent stem cells; Induced pluripotent stem cells; Uncharacterised stem cells
• 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 2500/14 Calcium; Ca chelators; Calcitonin
• CPC Class Codes: C12N 2501/999 Small molecules not provided for elsewhere
• CPC Class Codes: C12N 2502/13 connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
• CPC Class Codes: C12N 2506/04 from germ cells
• CPC Class Codes: C12N 2510/00 Genetically modified cells
• CPC Class Codes: C12N 2517/10 Conditioning of cells for in vitro fecondation or nuclear transfer
• 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
• CPC Class Codes: C12N 5/0696 Artificially induced pluripotent stem cells, e.g. iPS
• CPC Class Codes: G01N 2333/475 Assays involving growth factors
• CPC Class Codes: G01N 33/5008 for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
• CPC Class Codes: G01N 33/502 for testing non-proliferative effects
• CPC Class Codes: G01N 33/5044 involving specific cell types
• CPC Class Codes: G01N 33/5073 Stem cells
• CPC Class Codes: G01N 33/5088 of vertebrates

Abstract

Methods for obtaining pluripotent (embryonic stem) cells from parthenogenetic embryos, especially primates, are provided. These cells are useful for producing differentiated cells, tissues and organs, especially human and non-human primate cells, tissues and organs.

Claims

1. A method for producing pluripotent cells that can be used to produce differentiated cells in vitro comprising:

(a) obtaining an oocyte in metaphase II, which optionally may be genetically modified;

(b) activating said oocyte by a method selected from the group consisting of (1) culturing the oocyte under conditions that do not result in second polar body extrusion;

(2) culturing the oocyte in the presence of an agent that inhibits polar body extrusion, and (3) culturing the oocyte under conditions that prevent the initial cleavage;

(c) culturing said activated oocyte to produce a gynogenetic embryo comprising a discernable trophectoderm and an inner cell mass;

(d) isolating said inner cell mass or cells derived therefrom and transferring said inner cell mass or cells derived therefrom to an in vitro media that inhibits differentiation; and

(e) culturing said inner cell mass or cells derived therefrom to maintain said inner cell mass or cell derived therefrom in an undifferentiated pluripotent state;

wherein the activation conditions include use of a compound that inhibits microfilament or protein production.

2. The method of claim 1, wherein said compound is cycloheximide or cytochalasin B.

3. The method of claim 1, wherein the activated oocyte is incubated in DMAP for 3-4 hours.

4. The method of claim 1, wherein the oocyte is a human, non-human primate, bovine, porcine, or ovine oocyte.

5. The method of claim 1, wherein said cultured cells of (e) are allowed to differentiate.