Transformation of adult mesenchymal stem cells isolated from the fatty tissue into cardiomyocytes.
BACKGROUND
Myocardial infarction results in the death of cardiomyocytes, which are replaced by scar tissue. Cardiomyocytes cannot regenerate because they are terminally differentiated. Mesenchymal cells are pluripotent cells, which have the potential to differentiate to specialized tissues under appropriate stimuli. The aim of this study was to direct differentiation of the adult mesenchymal stem cells isolated from fatty tissue into cardiomyocytes using 5-azacytidine.


METHODS
Adult mesenchymal stem cells were isolated from the fatty tissue of New Zealand White rabbits and cultured in RPMI medium. Second-passaged mesenchymal cells were treated with various concentrations of 5-azacytidine and incubated for different intervals of time. The cells were plated in six-well dishes at 500, 5,000, and 50,000 cells/well. These cells were treated with 1-, 3-, 6-, 9-, and 12-micromol/L concentrations of 5-azacytidine and incubated for 12, 24, 48, and 72 hours. Later, the medium was replaced with fresh medium and incubated in a CO2 incubator. The medium was changed once at 3 to 4 days. At 2 months, the cells were fixed with 0.4% glutaraldehyde for 2 hours and later washed with phosphate-buffered saline. The transformed cells were subjected to immunostaining for the myosin heavy chain, alpha actinin, and troponin-I.


RESULTS
After treatment with 5-azacytidine, the adult mesenchymal stem cells were transformed into cardiomyocytes. At 1 week, some cells showed binucleation and extended cytoplasmic processes with adjacent cells. At 2 weeks, 20% to 30% of the cells increased in size and formed a ball-like appearance. At 3 weeks, these cells began to beat spontaneously in culture when observed under phase contrast microscope. Immunostaining of the transformed cells for myosin heavy chain, alpha actinin, and troponin-I was positive. The differentiated cells maintained the phenotype and did not dedifferentiate up to 2 months after treatment with 5-azacytidine.


CONCLUSIONS
These observations confirm that adult mesenchymal stem cells isolated from fatty tissue can be chemically transformed into cardiomyocytes. This can potentially be a source of autologous cells for myocardial repair.
Rangappa
Sunil
S
Division of Cardiothoracic Surgery, National University Hospital, National University of Singapore, Singapore. sunil_ran@hotmail.com
Fen
Chen
C
Lee
Eng Hin
EH
Bongso
Ariff
A
Sim
Eugene Kwang Wei
EK
Wei
Eugene Kwang Sim
EK
D000185:Actinin / Q000032:analysis; D000273:Adipose Tissue / Q000166:cytology*; D000818:Animals; D001374:Azacitidine / Q000494:pharmacology; D002454:Cell Differentiation / Q000502:physiology*; D007124:Immunoenzyme Techniques; D008648:Mesoderm / Q000166:cytology*; D009200:Myocardial Contraction / Q000502:physiology; D009206:Myocardium / Q000166:cytology*; D018995:Myosin Heavy Chains / Q000032:analysis; D011817:Rabbits; D013234:Stem Cells / Q000166:cytology*; D023822:Tissue Engineering / Q000379:methods*
D000185:Actinin; D018995:Myosin Heavy Chains; D001374:Azacitidine