Isolation of non-embryonic stem cells and uses thereof
AssigneeThe Jackson Laboratory
InventorWa Xian
Life of this patent
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Abstract
The invention described herein relates to methods of isolating non-embryonic stem cell, e.g., adult stem cell, from a non-embryonic tissue, e.g., an adult tissue or organ. Non-embryonic stem cells (e.g., adult stem cells) thus isolated from the various tissues or organs can self-renew or propagate indefinitely in vitro, are multipotent and can differentiate into the various differentiated cell types normally found within the tissue or organ from which the stem cells are isolated. In addition, the isolated stem cells can be propagated through clonal expansion of a single isolated stem cell, to produce a clone of which at least about 40%, 70%, or 90% or more cells within the clone can be further passaged as single cell originated clones.
Claims37
A method for isolating a non-embryonic stem cell from a non-embryonic tissue, the method comprising:
- (1) culturing dissociated epithelial cells from the non-embryonic tissue, in contact with a first population of lethally irradiated feeder cells and a basement membrane matrix, to form epithelial cell clones, in a medium comprising:
- (a) a Notch agonist;
- (b) a ROCK (Rho Kinase) inhibitor;
- (c) a Bone Morphogenetic Protein (BMP) antagonist;
- (d) a Wnt agonist;
- (e) a mitogenic growth factor;
- and,
- (f) insulin or IGF (or an agonist thereof);
- said medium optionally further comprising at least one of:
- (g) a TGFβ receptor inhibitor;
- and,
- (h) nicotinamide or an analog thereof;
- (2) isolating single cells from said epithelial cell clones, and,
- (3) culturing isolated single cells from step (2) individually to form single cell clones, in contact with a second population of lethally irradiated feeder cells and a second basement membrane matrix in said medium;
- wherein each of said single cell clones represents a clonal expansion of said non-embryonic stem cell, thereby isolating said non-embryonic stem cell.
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A single cell clone of a non-embryonic stem cell, wherein at least about 40%, 50%, 60%, 70%, or about 80% of cells within said single cell clone, when isolated as single cell, is capable of proliferation to produce single cell clone.
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A single cell clone of a non-embryonic stem cell, wherein said non-embryonic stem cell expresses one or more stem cell markers selected from SOX9, KRT19, KRT7, LGR5, CA9, FXYD2, CDH6, CLDN18, TSPAN8, BPIFB1, OLFM4, CDH17, and PPARGC1A, such as a small intestine stem cell, which expresses one or more markers selected from:
- OLFM4, SOX9, LGR5, CLDN18, CA9, BPIFB1, KRT19, CDH17, and TSPAN8.
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A medium for isolating and/or culturing non-embryonic stem cell, said medium comprising:
- (a) a Notch agonist;
- (b) a ROCK (Rho Kinase) inhibitor;
- (c) a Bone Morphogenetic Protein (BMP) antagonist;
- (d) a Wnt agonist;
- (e) a mitogenic growth factor;
- and,
- (f) insulin or IGF,
- and optionally may also include:
- (g) a TGFβ receptor inhibitor;
- and,
- (h) nicotinamide or a precursor thereof.
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Dependencies are inferred from the claim text.