Differential regulation of smooth muscle markers in human bone marrow-derived mesenchymal stem cells.
OBJECTIVE
To study smooth-muscle differentiation and de-differentiation of human bone marrow-derived mesenchymal stem cells (MSCs), which have been shown to enter the circulation and to contribute to vascular repair and atherosclerosis.


METHODS
Human MSCs from bone marrow were cultured with 20% fetal calf serum (FCS) or with 10% FCS and various concentrations of dimethyl sulfoxide (DMSO). Expression of smooth muscle markers was determined by Western blot analysis and immunofluorescence. For signalling studies, involvement of the mammalian target of rapamycin (mTOR) pathway was tested by treatment with rapamycin.


RESULTS
MSCs cultured with 20% FCS acquired a smooth muscle-like appearance and expressed the smooth muscle (sm) markers sm-alpha-actin, desmin, sm-calponin and myosin light chain kinase (MLCK). DMSO induced a spindle-like morphology with marked reduction of stress fibers. As judged by Western blot analysis, treatment with 2.5% DMSO strongly downregulated expression of sm-calponin (-85%), short MLCK (-98%) and sm-alpha-actin expression (-51%). Reduced calponin expression was detected by day 2 of treatment with 0.5-2.5% DMSO. After withdrawal of DMSO, MSCs regained high expression of sm-calponin. Treatment with 6 nmol/l rapamycin partly antagonized the effect of DMSO, indicating the involvement of mTOR in regulation of the smooth muscle phenotype of MSCs.


CONCLUSIONS
DMSO strongly downregulates the smooth muscle markers sm-calponin, short MLCK and sm-alpha-actin in human MSCs, indicating a transition from a smooth muscle-like phenotype to an undifferentiated state by an mTOR-dependent mechanism. Regulating the phenotype of human MSCs may be of relevance for novel therapeutic approaches in atherosclerosis and intimal hyperplasia after vascular injury.
Hegner
Björn
B
Department of Medicine, Merheim Medical Center, Cologne General Hospital, Germany.
Weber
Manfred
M
Dragun
Duska
D
Schulze-Lohoff
Eckhard
E
D000199:Actins / Q000378:metabolism; D000328:Adult; D000368:Aged; D000903:Antibiotics, Antineoplastic / Q000494:pharmacology; D015415:Biomarkers; D001854:Bone Marrow Cells / Q000166:cytology* / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000378:metabolism; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology; D019070:Cell Lineage / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D005260:Female; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000378:metabolism*; D006801:Humans; D008297:Male; D008648:Mesoderm / Q000166:cytology; D008840:Microfilament Proteins; D008875:Middle Aged; D009124:Muscle Proteins / Q000378:metabolism; D009131:Muscle, Smooth, Vascular / Q000166:cytology* / Q000378:metabolism*; D009219:Myosin-Light-Chain Kinase / Q000378:metabolism; D010641:Phenotype; D011494:Protein Kinases / Q000378:metabolism; D020123:Sirolimus / Q000494:pharmacology; D012997:Solvents / Q000494:pharmacology; D058570:TOR Serine-Threonine Kinases; D000096985:Calponins
D000199:Actins; D000903:Antibiotics, Antineoplastic; D015415:Biomarkers; D002135:Calcium-Binding Proteins; D004121:Dimethyl Sulfoxide; D008840:Microfilament Proteins; D009124:Muscle Proteins; D009219:Myosin-Light-Chain Kinase; D011494:Protein Kinases; D020123:Sirolimus; D012997:Solvents; D058570:TOR Serine-Threonine Kinases; D000096985:Calponins; C546842:MTOR protein, human