De-differentiation and re-differentiation of somatic cells and production of cells for cell therapies
The invention provides a method for effecting the de-differentiation of a somatic cell by culturing the cell in the absence of growth factors, cytokines, or other differentiation-inducing agents, and introducing components of cytoplasm of plutipotent cells into the somatic cell and allowing the cell to de-differentiate. The method can be used with somatic cells of any type, from any species of animal. The pluripotent cells may be oocytes, blastomeres, inner cell mass cells, embryonic stem cells, embryonic germ cells, embryos consisting of one or more cells, embryoid body (embryoid) cells, moruia-derived cells, teratoma (teratocarcinoma) cells, as well as multipotent partially differentiated embryonic stem cells taken from later in the embryonic development process. After being de-differentiated, the cell can be induced to re-differentiate into a different somatic cell type. A method for de-differentiating a somatic cell and inducing it to re-differentiate into a cell of neural lineage is disclosed.
1 . A method for effecting de-differentiation of a somatic cell comprising
(a) culturing a somatic cell in the absence of growth factors, cytokines, or other differentiation-inducing agents,
(b) introducing components of cytoplasm of plutipotent cells into the somatic cell; and
(c) allowing the cell to de-differentiate.
2 . The method of claim 1 , wherein the cell is a mammalian somatic cell selected from the group consisting of fibroblasts, B cells, T cells, dendritic cells, keratinocytes, adipose cells, epithelial cells, epidermal cells, chondrocytes, cumulus cells, neural cells, glial cells, astrocytes, cardiac cells, esophageal cells, muscle cells, melanocytes, hematopoietic cells, macrophages, monocytes, and mononuclear cells.
3 . The method of claim 1 , wherein step (a) comprises culturing the cell in serum-free medium.
4 . The method of claim 1 , wherein the pluripotent cells are selected from the group consisting of oocytes, blastomeres, inner cell mass cells, embryonic stem cells, embryonic germ cells, embryos consisting of one or more cells, embryoid body (embryoid) cells, morula-derived cells, teratoma (teratocarcinoma) cells, as well as multipotent partially differentiated embryonic stem cells taken from later in the embryonic development process.
5 . The method of claim 1 , wherein the pluripotent cells are oocytes.
6 . The method of claim 5 , wherein the oocytes are metaphase II oocytes.
7 . The method of claim 5 , wherein the oocytes are Xenopus oocytes.
8 . The method of claim 1 , further comprising centrifuging oocyte cytoplasm, and isolating a fraction of the centrifuged oocyte cytoplasm containing the components of cytoplasm of step (b).
9 . The method of claim 1 , wherein step (b) comprises placing the somatic cell in solution containing components of cytoplasm of pluripotent cells, and introducing components of cytoplasm of plutipotent cells into the somatic cell by electroporation.
10 . The method of claim 1 , further comprising, after the step of introducing components of cytoplasm of plutipotent cells,
culturing the cell under conditions suitable for maintaining pluripotent stem cells in an undifferentiated state
11 . The method of claim 1 , further comprising, after the step of introducing components of cytoplasm of plutipotent cells,
culturing the cell under conditions that induce or direct partial or complete differentiation to a particular cell type
12 . The method of claim 12 , comprising, after the step of introducing components of cytoplasm of plutipotent cells,
culturing the cell in medium containing nerve growth factor.
13 . The method of claim 12 , comprising, after the step of introducing components of cytoplasm of plutipotent cells,
culturing the cell in DMEM/F12 ITS medium that contains nerve growth factor.
14 . A method for reprogramming a somatic cell to become a cell of neural lineage, comprising:
(a) culturing a somatic cell that is not of neural lineage in the absence of growth factors, cytokines, or other differentiation-inducing agents,
(b) introducing cytoplasm of a pluripotent cell into the cell; and
(c) culturing the cell in medium containing nerve growth factor.
15 . The method of claim 14 , wherein step (c) comprises culturing the cell in DMEM/F12 ITS medium that contains nerve growth factor.
16 . The method of claim 14 , further comprising assaying to detect a marker of cells of neural lineage.
17 . A composition of cells of neural lineage prepared by the method of claim 14.