Multiple mesodermal lineage differentiation potentials for adipose tissue-derived stromal cells and uses thereof
OwnerJeffrey Martin Gimble, Yuan-Di Chang Halvorsen +1inventors
InventorsJeffrey Martin Gimble, Yuan-Di Chang Halvorsen, William O. Wilkison
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Abstract
The invention relates to methods and compositions for the differentiation of stromal cells from adipose tissue into hematopoietic supporting stromal cells and myocytes of both the skeletal and smooth muscle type. The cells produced by the methods are useful in providing a source of fully differentiated and functional cells for research, transplantation and development of tissue engineering products for the treatment of human diseases and traumatic tissue injury repair.
Claims79
A medium for differentiating adipose tissue derived stromal cells into hematopoietic supporting stromal cells that will proliferate and differentiate along the myeloid lineage pathway or the B-lineage lymphoid pathway, said medium comprising:
- a chemically defined medium having or supplemented with the following components present in sufficient amounts to stimulate differentiation (i) 0% to 20% fetal bovine serum (ii) antibiotic (iii) interleukins (iv) stem cell factor (v) flt-3 ligand (vi) macrophage-colony stimulating factor (vii) granulocyte-monocyte colony stimulating factor (viii) erythropoietin (ix) thrombopoietin (x) osteoprotegerin ligand (xi) dexamethasone (xii) hydrocortisone (xiii) 1,25 dihydroxy vitamin D3 and (xiv) 2-mercaptoethanol.
A medium for differentiating adipose tissue derived stromal cells into skeletal muscle myocytes, said medium comprising:
- a chemically defined medium having or supplemented with the following components present in sufficient amounts to stimulate differentation (i) 0.5% to 20% fetal bovine serum (ii) an antibiotic (iii) glutamine (iv) sodium pyruvate (v) 2-mercaptoethanol and (vi) 5′ azacytadine or amphotericin.
A medium for differentiating adipose tissue derived stromal cells into smooth muscle myoblasts, said medium comprising:
- a chemically defined medium having or supplemented with the following components present in amounts sufficient to stimulate differentiation (i) 0% to 10% fetal bovine serum (ii) an antibiotic (iii) glutamine (iv) sodium pyruvate (v) transforming growth factor β or fibroblast growth factor and (vi) a 3-dimensional matrix composed of a biodegradable material.
A method for differentiating adipose tissue derived stromal cells into hematopoietic supporting stromal cells which will proliferate and differentiate along the myeloid lineage pathway or the B-lineage lymphoid pathway, comprising:
- a) plating said stromal cells at a density of about 30,000 cells per cm2 in chamber slides;
- b) maintaining cells in culture for about 8 days in a medium containing Dulbecco's Modified Eagle's Medium (DMEM) or Ham's F-10;
- c) supplementing said medium with:
- (i) 1 to 20% fetal bovine serum
- (ii) an antibiotic
- (iii) interleukins (iv) stem cell factor (v) flt-3 ligand (vi) macrophage-colony stimulating factor (vii) granulocyte-monocyte colony stimulating factor (viii) erythropoetin (ix) thrombopoietin (x) osteoprotegerin ligand (xi) dexamethasone (xii) hydrocortisone (xiii) 1,25 dihydroxy vitamin D3 and (xiiii) 2-mercaptoethanol;
- and
- d) examining the expression of cell surface proteins which are consistent with cells of the myeloid lineage or B-lymphoid lineage using a variety of techniques which include but are not limited to immunohistochemistry, flow cytometry, immunofluorescence and mRNA expression in cell populations.
A method for differentiating adipose tissue derived stromal cells into skeletal muscle myocyte cells, comprising:
- a) plating said stromal cells at a density of about 500 to about 20,000 cells per cm2 in chamber slides;
- b) maintaining cells in culture for about 8 days in a medium containing Dulbecco's Modified Eagle's Medium (DMEM) or Ham's F-10;
- c) supplementing said medium with:
- (i) 1 to 10% fetal bovine serum
- (ii) an antibiotic
- (iii) glutamine (iv) sodium pyruvate (v) 2-mercaptoethanol;
- d) exposing cells to azacytadine or amphotericin for 1 to 6 days;
- e) examining said cells for biochemical phenotypes or markers characteristic of skeletal muscle myoblasts.
A method for differentiating adipose tissue derived stromal cells into smooth muscle myoblast cells, comprising:
- a) plating said stromal cells at a density of about 500 to about 20,000 cells per cm2 in chamber slides;
- b) maintaining cells in a medium containing Dulbecco's Modified Eagle's Medium (DMEM) or Ham's F-10;
- c) supplementing said medium with:
- (i) 1 to 10% fetal bovine serum
- (ii) an antibiotic
- (iii) glutamine (iv) sodium pyruvate (v) transforming growth factor β and/or fibroblast growth factor;
- d) maintaining cells as a monolayer or in a 3-dimensional lattice comprising collagen type I, or alginate or other biodegradable material;
- and
- e) characterizing cells for biochemical or functional criteria to establish smooth muscle differentiation.
Dependencies are inferred from the claim text.