Multiple mesodermal lineage differentiation potentials for adipose tissue-derived stromal cells and uses thereof

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.

That which is claimed:  
     
         1 . 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 D 3  and (xiv) 2-mercaptoethanol.  
     
     
         2 . The medium of  claim 1 , wherein said chemically defined medium is selected from the group consisting of: DMEM, αMEM, and RPMI media 1640.  
     
     
         3 . The medium of  claim 1 , wherein said antibiotic is penicillin.  
     
     
         4 . The medium of  claim 1 , wherein said antibiotic is streptomycin.  
     
     
         5 . The medium of  claim 3 , wherein said penicillin is present in amounts from about 10 units per ml to about 200 units per ml.  
     
     
         6 . The medium of  claim 4 , wherein said streptomycin is present in amounts from about 10 μg per ml to about 200 μg per ml.  
     
     
         7 . The medium of  claim 1 , wherein said interleukins are selected from the group consisting of: interleukin-1, interleukin-3, interleukin-6, interleukin-7, interleukin-11 and interleukin-12.  
     
     
         8 . The medium of  claim 7 , wherein the amount of interleukins is from about 5 pg/ml to about 1 ng/ml.  
     
     
         9 . The medium of  claim 1 , wherein said flt-3 ligand is present at amounts from about 5 pg/ml to about 1 ng/ml.  
     
     
         10 . The medium of  claim 1 , wherein said stem cell factor is present at amounts from about 5 pg/ml to about 1 ng/ml.  
     
     
         11 . The medium of  claim 1 , wherein said granulocyte-monocyte colony stimulating factor is present at amounts from about 5 pg/ml to about 1 ng/ml.  
     
     
         12 . The medium of  claim 1 , wherein said macrophage-colony stimulating factor is present at amounts from about 5 pg/ml to about 1 ng/ml.  
     
     
         13 . The medium of  claim 1 , wherein said erythropoietin is present at amounts from about 5 units/ml to about 1000 units/ml.  
     
     
         14 . The medium of  claim 1 , wherein said thrombopoietin is present at amounts from about 5 pg/ml to about 1 ng/ml.  
     
     
         15 . The medium of  claim 1 , wherein said osteoprotegerin ligand is present from about 5 pg/ml to about 1 ng/ml.  
     
     
         16 . The medium of  claim 1 , wherein said dexamethasone is present from about 1 nM to about 100 nM.  
     
     
         17 . The medium of  claim 1 , wherein said hydrocortisone is present from about 1 nM to about 100 nM.  
     
     
         18 . The medium of  claim 1 , wherein said 1,25 dihydroxy vitamin D 3  is present in amounts from about 1 nM to about 100 nM.  
     
     
         19 . The medium of  claim 1 , wherein said 2-mercaptoethanol is present in amounts from about 10 uM to about 100 uM.  
     
     
         20 . The medium of  claim 1 , wherein said medium is incubated at a temperature of about 33° C. for myeloid cells.  
     
     
         21 . The medium of  claim 1 , wherein said medium is incubated at a temperature of about 37° C. for B-lineage lymphoid cells.  
     
     
         22 . 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.  
     
     
         23 . The medium of  claim 22 , wherein said chemically defined medium is selected from the group consisting of DMEM, αMEM, and RPMI media 1640.  
     
     
         24 . The medium of  claim 22 , wherein said antibiotic is penicillin.  
     
     
         25 . The medium of  claim 22 , wherein said antibiotic is streptomycin.  
     
     
         26 . The medium of  claim 24 , wherein said penicillin is present in amounts from about 10 to about 200 units per ml.  
     
     
         27 . The medium of  claim 25 , wherein said streptomycin is present in amounts from about 10 μg per ml to about 200 μg per ml.  
     
     
         28 . The medium of  claim 22 , wherein said sodium pyruvate is present from about 0.5 mM to about 2 mM.  
     
     
         29 . The medium of  claim 22 , wherein said 2-mercaptoethanol is present from about 10 μM to about 100 μM.  
     
     
         30 . The medium of  claim 22 , wherein said 5′ azacytadine is present in amounts sufficient to induce the differentiation of said stromal cells into skeletal muscle myocytes.  
     
     
         31 . The medium of  claim 30 , wherein said 5′ azacytadine is present from about 1 μM to about 30 μM.  
     
     
         32 . The medium of  claim 22 , wherein said amphotericin is present in amounts sufficient to induce the differentiation of said stromal cells into skeletal muscle myocytes.  
     
     
         33 . The medium of  claim 32 , wherein said amphotericin is present from about 10 ng per ml to about 100 ng per ml.  
     
     
         34 . 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.  
     
     
         35 . The medium of  claim 34 , wherein said chemically defined medium is selected from the group consisting of: DMEM, αMEM, and RPMI media 1640.  
     
     
         36 . The medium of  claim 34 , wherein said antibiotic is penicillin.  
     
     
         37 . The medium of  claim 34 , wherein said antibiotic is streptomycin.  
     
     
         38 . The medium of  claim 36 , wherein said penicillin is present from about 10 units per ml to about 200 units per ml.  
     
     
         39 . The medium of  claim 37 , wherein said streptomycin is present from about 10 μg per ml to about 200 μg per ml.  
     
     
         40 . The medium of  claim 34 , wherein said sodium pyruvate is present from about 0.5 to about 2 mM.  
     
     
         41 . The medium of  claim 34 , wherein said transforming growth factor β is present from about 20 ng per ml to about 40 ng per ml.  
     
     
         42 . The medium of  claim 34 , wherein said fibroblast growth factor is present in amounts from about 20 ng per ml to about 40 ng per ml.  
     
     
         43 . The medium of  claim 34 , wherein the biodegradable material is selected from the group comprising collagen type I, alginate, or other synthetic type polymer.  
     
     
         44 . 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 cm 2  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 D 3  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.    
     
     
         45 . The medium of  claim 44 , wherein said antibiotic is penicillin.  
     
     
         46 . The medium of  claim 44 , wherein said antibiotic is streptomycin.  
     
     
         47 . The medium of  claim 45 , wherein said penicillin is present in amounts from about 10 to about 200 units per ml.  
     
     
         48 . The medium of  claim 46 , wherein said streptomycin is present in amounts from about 10 μg per ml to about 200 μg per ml.  
     
     
         49 . The medium of  claim 44 , wherein said interleukins are selected from the group consisting of: interleukin-1, interleukin-3, interleukin-6, interleukin-7, interleukin-11 and interleukin-12.  
     
     
         50 . The medium of  claim 49 , wherein the amount of interleukins is present in amounts from about 5 pg/ml to about 1 ng/ml.  
     
     
         51 . The medium of  claim 44 , wherein said flt-3 ligand is present at amounts from about 5 pg/ml to about 1 ng/ml.  
     
     
         52 . The medium of  claim 44 , wherein said stem cell factor is present in amounts from about 5 pg/ml to about 1 ng/ml.  
     
     
         53 . The medium of  claim 44 , wherein said granulocyte-monocyte colony stimulating factor is present in amounts from about 5 pg/ml to about 1 ng/ml.  
     
     
         54 . The medium of  claim 44 , wherein said macrophage-colony stimulating factor is present at amounts from about 5 pg/ml to about 1 ng/ml.  
     
     
         55 . The medium of  claim 44 , wherein said erythropoietin is present at amounts from about 5 units/ml to about 1000 units/ml.  
     
     
         56 . The medium of  claim 44 , wherein said thrombopoietin is present at amounts from about 5 pg/ml to about 1 ng/ml.  
     
     
         57 . The medium of  claim 44 , wherein said osteoprotegerin ligand is present from about 5 pg/ml to about 1 ng/ml.  
     
     
         58 . The medium of  claim 44 , wherein said dexamethasone is present from about 1 nM to about 100 nM.  
     
     
         59 . The medium of  claim 44 , wherein said hydrocortisone is present from about 1 nM to about 100 nM.  
     
     
         60 . The medium of  claim 44 , wherein said 1,25 dihydroxy vitamin D 3  is present in amounts from about 1 to about 10 nM.  
     
     
         61 . The medium of  claim 1 , wherein the 2-mercaptoethanol is present from about 10 μM to about 100 μM.  
     
     
         62 . 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 cm 2  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.    
     
     
         63 . The method according to  claim 62 , wherein said antibiotic is penicillin.  
     
     
         64 . The method according to  claim 62 , wherein said antibiotic is streptomycin.  
     
     
         65 . The method according to  claim 63 , wherein said penicillin is present from about 10 units per ml to about 200 units per ml.  
     
     
         66 . The method according to  claim 64 , wherein said streptomycin is present from about 10 μg per ml to about 200 μg per ml.  
     
     
         67 . The method according to  claim 62 , wherein said sodium pyruvate is present from about 0.5 mM to about 2 mM.  
     
     
         68 . The method according to  claim 62 , wherein said 2-mercaptoethanol is present from about 10 μM to about 100 μM.  
     
     
         69 . The method according to  claim 62 , wherein said 5′ azacytadine is present from about 1 μM to about 30 μM.  
     
     
         70 . The method according to  claim 62 , wherein said amphotericin is present from about 10 ng/ml to about 100 ng/ml.  
     
     
         71 . 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 cm 2  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.    
     
     
         72 . The method according to  claim 71 , wherein said antibiotic is penicillin.  
     
     
         73 . The method according to  claim 71 , wherein said antibiotic is streptomycin.  
     
     
         74 . The method according to  claim 72 , wherein said penicillin is present from about 10 units per ml to about 200 units per ml.  
     
     
         75 . The method according to  claim 73 , wherein said streptomycin is present from about 10 μg per ml to about 200 ug per ml.  
     
     
         76 . The method according to  claim 71 , wherein said sodium pyruvate is present from about 0.5 mM to about 2 mM.  
     
     
         77 . The method according to  claim 71 , wherein said transforming growth factor β is present from about 20 ng/ml to about 40 ng/ml.  
     
     
         78 . The method according to  claim 71 , wherein said fibroblast growth factor is present from about 20 ng/ml to about 40 ng/ml.  
     
     
         79 . A hematopoietic cell made by the method of  claim 44.