Differentiation of smooth muscle progenitor cells in peripheral blood and its application in tissue engineered blood vessels.
BACKGROUND
A major shortcoming in tissue engineered blood vessels (TEBVs) is the lack of healthy and easily attainable smooth muscle cells (SMCs). Smooth muscle progenitor cells (SPCs), especially from peripheral blood, may offer an alternative cell source for tissue engineering involving a less invasive harvesting technique.


METHODS
SPCs were isolated from 5-ml fresh rat peripheral blood by density-gradient centrifugation and cultured for 3 weeks in endothelial growth medium-2-MV (EGM-2-MV) medium containing platelet-derived growth factor-BB (PDGF BB). Before seeded on the synthesized scaffold, SPC-derived smooth muscle outgrowth cell (SOC) phenotypes were assessed by immuno-fluorescent staining, Western blot analysis, and reverse transcription polymerase chain reaction (RT-PCR). The cells were seeded onto the silk fibroin-modified poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (SF-PHBHHx) scaffolds by 6x10(4) cells/cm2 and cultured under the static condition for 3 weeks. The growth and proliferation of the seeded cells on the scaffold were analyzed by 3-(4,5-dimethylthiazol-2-yl)-diphenyltetrazolium bromide (MTT) assay, scanning electron microscope (SEM), and 4,6-diamidino-2-phenylindole (DAPI) staining.


RESULTS
SOCs displayed specific "hill and valley" morphology, expressed the specific markers of the SMC lineage: smooth muscle (SM) alpha-actin, calponin and smooth muscle myosin heavy chain (SM MHC) at protein and messenger ribonucleic acid (mRNA) levels. RT-PCR results demonstrate that SOCs also expressed smooth muscle protein 22alpha (SM22alpha), a contractile protein, and extracellular matrix components elastin and matrix Gla protein (MGP), as well as vascular endothelial growth factor (VEGF). After seeded on the SF-PHBHHx scaffold, the cells showed excellent metabolic activity and proliferation.


CONCLUSIONS
SPCs isolated from peripheral blood can be differentiated into the SMCs in vitro and have an impressive growth potential in the biodegradable synthesized scaffold. Thus, SPCs may be a promising cell source for constructing TEBVs.
Xie
Shang-zhe
SZ
Laboratory of Molecular Biology, Shanghai Medical College, Fudan University, Shanghai 200032, China.
Fang
Ning-tao
NT
Liu
Shui
S
Zhou
Ping
P
Zhang
Yi
Y
Wang
Song-mei
SM
Gao
Hong-yang
HY
Pan
Luan-feng
LF
D020155:3-Hydroxybutyric Acid / Q000737:chemistry; D000818:Animals; D001808:Blood Vessels / Q000166:cytology*; D002208:Caproates / Q000737:chemistry; D002448:Cell Adhesion; D002454:Cell Differentiation; D049109:Cell Proliferation; D016130:Immunophenotyping; D008855:Microscopy, Electron, Scanning; D009131:Muscle, Smooth, Vascular / Q000166:cytology*; D032389:Myocytes, Smooth Muscle / Q000166:cytology*; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D023822:Tissue Engineering; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics; D000094122:Polyhydroxybutyrates
D020155:3-Hydroxybutyric Acid; D002208:Caproates; D012333:RNA, Messenger; D042461:Vascular Endothelial Growth Factor A; C115940:poly(3-hydroxybutyrate-co-3-hydroxyhexanoate); D000094122:Polyhydroxybutyrates