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.

1 .- 70 . (canceled) 
     
     
         71 . A method for differentiating adipose tissue derived stromal cells into cells that express at least one characteristic of a myoblast, 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 myoblast 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 . (canceled) 
     
     
         80 . The method according to  claim 71 , wherein the at least one characteristic of a myoblast is the expression of muscle heavy chain myosin kinase. 
     
     
         81 . The method according to  claim 71 , wherein the at least one characteristic of a myoblast is the expression of muscle light chain myosin kinase. 
     
     
         82 . The method according to  claim 71 , wherein the at least one characteristic of a myoblast is the expression of myosin. 
     
     
         83 . The method according to  claim 71 , wherein the at least one characteristic of a myoblast is the expression of skeletal muscle actin. 
     
     
         84 . The method according to  claim 71 , wherein the at least one characteristic of a myoblast is the expression of myogenic transcription factors. 
     
     
         85 . The method according to  claim 71 , wherein the myogenic transcription factor is selected from myoD and myogenin. 
     
     
         86 . The method according to  claim 71 , wherein the at least one characteristic of a myoblast is the expression of smooth muscle actin. 
     
     
         87 . The method according to  claim 71 , wherein the myoblast is a skeletal muscle myoblast. 
     
     
         88 . The method according to  claim 71 , wherein the myoblast is a smooth muscle myoblast. 
     
     
         89 . The method according to  claim 71 , wherein the myoblast is a cardiac muscle myoblast.