Control of es cell self renewal and lineage specification, and medium therefor
OwnerAustin Smith, Qi-Long Ying +1inventors
InventorsAustin Smith, Qi-Long Ying, Jennifer Nichols
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Abstract
Self renewal of pluripotent cells in culture is promoted using a combination of an Id gene product and an activator of a gp130 downstream signalling pathway.
Claims38
A method of promoting self-renewal of pluripotent cells in culture, comprising culturing pluripotent cells in a medium comprising an Id gene product.
(canceled)
A method of promoting self-renewal of a pluripotent cell in culture in medium that is free of serum and free of serum extract, comprising (1) expressing an Id gene or inducing expression of an Id gene in the cell, or culturing the cell in medium containing an Id protein, and (2) activating gp130 downstream signalling.
A method of promoting self-renewal of a pluripotent cell in culture, comprising culturing a pluripotent cell in a medium comprising:
- (a) a direct activator or effector of Id gene expression and/or Id protein activity, other than one acting through a receptor of the TGF-β superfamily;
- and
- (b) an activator of a gp130 downstream signalling pathway, wherein the medium is free of serum and free of serum extract.
A method of culture of ES cells to promote ES cell self renewal in medium that is free of serum and free of serum extract, comprising maintaining the ES cells in medium comprising:
- (a) an Id protein or a direct activator or effector of Id gene expression and/or Id protein activity, other than one acting through a receptor of the TGF-β superfamily;
- and
- (b) an activator of a gp130 downstream signalling pathway.
A method of culture of ES cells, comprising the steps of:
- (a) maintaining the ES cells in a pluripotent state in culture, optionally on feeders feeder cells, in the presence of a cytokine acting through gp130 and serum or an extract of serum;
- (b) passaging the ES cells at least once;
- (c) withdrawing the serum or the serum extract from the medium and withdrawing the feeder cells if present, so that the medium is free of feeder cells, serum and serum extract;
- and
- (d) subsequently maintaining ES cells in a pluripotent state by culturing the cells in a medium comprising:
- (i) a direct activator or effector of Id gene expression and/or Id protein activity, other than one acting through the receptor of the TGF-β superfamily;
- and
- (ii) an activator of a gp130 downstream signalling pathway.
A method of obtaining a transfected population of ES cells, comprising the steps of:
- (a) transfecting ES cells with a construct encoding a selectable marker operably linked to a promoter that expresses the selectable marker preferentially in an ES cell:
- (b) plating the ES cells;
- (c) culturing the ES cells in the presence of
- (i) a direct activator or effector of Id gene expression and/or Id protein activity, other than one activator acting through a receptor of the TGF-β superfamily;
- and
- (ii) an activator of a gp130 downstream signalling pathway;
- and
- (d) selecting for cells that express the selectable marker.
A method of culture of ES cells in medium that is free of serum and free of serum extract, comprising transferring an individual ES cell to a culture vessel and culturing the ES cell in the presence of
- (a) a direct activator or effector of Id gene expression and/or Id protein activity, other than one acting through a receptor of the TGF-β superfamily;
- and
- (b) an activator of a gp130 downstream signalling pathway, so as to obtain a clonal population of ES cells, all of which are progeny of a single ES cell.
A medium for self-renewal of ES cells, comprising:
- (a) basal medium;
- (b) a direct activator or effector of Id gene expression and/or Id protein activity, other than one acting through a receptor of the TGF-β superfamily;
- (c) an activator of gp130 downstream signalling pathways;
- and
- (d) an iron transporter,
- wherein the medium is free of serum or serum extract.
A method of promoting self-renewal of pluripotent cells in culture, comprising culturing pluripotent cells in a medium comprising an agent that increases Id protein activity, wherein the medium free of serum and free of serum extract.
A method of obtaining a pluripotent cell in medium that is free of serum and free of serum extract, comprising
- expressing an Id gene or inducing expression of an Id gene in a cell, or culturing a cell in medium containing an Id protein, and activating gp130 downstream signalling in the cell, wherein the cell is obtained from somatic cells or tissue of a fetus or adult.
A method of promoting self-renewal of pluripotent cells in culture, comprising culturing pluripotent cells in a medium comprising:
- (a) an agent that increases Id gene expression or activity;
- and
- (b) an activator of a gp130 downstream signalling pathway, wherein the medium is free of serum and free of serum extract.
Dependencies are inferred from the claim text.