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.

What is claimed is: 
     
         1 . 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 (xiv) 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.   
     
     
         2 . The method of  claim 1 , wherein said antibiotic is penicillin. 
     
     
         3 . The method of  claim 1 , wherein said antibiotic is streptomycin. 
     
     
         4 . The method of  claim 2 , wherein said penicillin is present in amounts from about 10 to about 200 units per ml. 
     
     
         5 . The method of  claim 3 , wherein said streptomycin is present in amounts from about 10 μg per ml to about 200 μg per ml. 
     
     
         6 . The method 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. 
     
     
         7 . The method of  claim 6 , wherein the amount of interleukins is present in amounts from about 5 pg/ml to about 1 ng/ml. 
     
     
         8 . The method of  claim 1 , wherein said flt-3 ligand is present at amounts from about 5 pg/ml to about 1 ng/ml. 
     
     
         9 . The method of  claim 1 , wherein said stem cell factor is present in amounts from about 5 pg/ml to about 1 ng/ml. 
     
     
         10 . The method of  claim 1 , wherein said granulocyte-monocyte colony stimulating factor is present in amounts from about 5 pg/ml to about 1 ng/ml. 
     
     
         11 . The method of  claim 1 , wherein said macrophage-colony stimulating factor is present at amounts from about 5 pg/ml to about 1 ng/ml. 
     
     
         12 . The method of  claim 1 , wherein said erythropoietin is present at amounts from about 5 units/ml to about 1000 units/ml. 
     
     
         13 . The method of  claim 1 , wherein said thrombopoietin is present at amounts from about 5 pg/ml to about 1 ng/ml. 
     
     
         14 . The method of  claim 1 , wherein said osteoprotegerin ligand is present from about 5 pg/ml to about 1 ng/ml. 
     
     
         15 . The method of  claim 1 , wherein said dexamethasone is present from about 1 nM to about 100 nM. 
     
     
         16 . The method of  claim 1 , wherein said hydrocortisone is present from about 1 nM to about 100 nM. 
     
     
         17 . The method of  claim 1 , wherein said 1,25 dihydroxy vitamin D 3  is present in amounts from about 1 to about 10 nM. 
     
     
         18 . The method of  claim 1 , wherein the 2-mercaptoethanol is present from about 10 μM to about 100 μM.