[Experiment of adipose derived stem cells induced into smooth muscle cells].
OBJECTIVE
To study the feasibility of human adipose derived stem cells (ADSCs) in monolayer culture induced into smooth muscle cells in vitro as seeding cells in vascular tissue engineering.


METHODS
The mononuclear cells in human adipose were separated by collagenase treatment and seeded on culture dishes with the density of 5 x 10(5)/cm2. Cells were cultured in M-199 plus 10% FBS. When reaching confluence, the cells were subcultured by 0.1% trypsin and 0.02% EDTA treatment, PDGF-BB (50 ng/mL) and TGF-beta1 (5 ng/mL) were added at the passage 1 to enhance the smooth muscle cells' phenotype. Cells were cultured under the inducing medium for 14 days. The morphology of induced cells was observed under the microscope. Cellular immunofluorescence and RT-PCR were used to determine the expression of smooth muscle cell markers of the post-induced cells. Flow cytometry (FACs) was used to examine the positive rate of induced team.


RESULTS
Cocultured in M-199 media including TGF-beta1 and PDGF-BB, the proliferating capability of the induced cells was significantly downregulated compared with the uninduced cells (P < 0.01). The induced cells exhibited "Hill and Valley" morphology, while the uninduced cells were similar to ADSCs of P0 which had the fibroblast-like morphology. The results of immunofluorescence indicated that the induced cells expressed smoothmuscle (SM) cell-specific markers including a-smooth muscle actin (alpha-SMA), SM-myosin heavy chain (SM-MHC) and Calponin. The results of RT-PCR revealed that the induced cells also expressed alpha-SMA, SM-MHC, Calponin and SM-22alpha. The positive rates of alpha-SMA, SM-MHC and Calponin in FACs were 3.26% +/- 1.31%, 3.55% +/- 1.6% and 4.02% +/- 1.81%, respectively, before the cells were induced. However, 14 days after the cell induction, the positive rates were 48.13% +/- 8.31%, 45.33% +/- 10.68% and 39.13% +/- 9.42%, respectively. The positive rates in induced cells were remarkably higher than those in uninduced cells (P < 0.01).


CONCLUSIONS
The human ADSCs can be induced to express vascular smooth muscle markers, and they are a new potential source of vascular tissue engineering.
Department of Plastic and Reconstructive Surgery, Ninth People's Hospital, Affiliated to Shanghai Jiaotong University, Shanghai, 200011, P.R. China.
Ping
P
Yang
Shuo
S
Yin
Lei
L
Cui
Hong
H
Li
Yingchen
Y
Wu
Wei
W
Liu
Yilin
Y
Cao
D000199:Actins / Q000096:biosynthesis; D000273:Adipose Tissue / Q000166:cytology*; D000077214:Becaplermin; D001807:Blood Vessel Prosthesis; D002454:Cell Differentiation / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D005434:Flow Cytometry; D006801:Humans; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000378:metabolism; D009131:Muscle, Smooth, Vascular / Q000166:cytology* / Q000378:metabolism; D032389:Myocytes, Smooth Muscle / Q000166:cytology / Q000378:metabolism; D018995:Myosin Heavy Chains / Q000378:metabolism; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology; D020574:Proto-Oncogene Proteins c-sis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D023822:Tissue Engineering; D053773:Transforming Growth Factor beta1 / Q000494:pharmacology
D000199:Actins; D010982:Platelet-Derived Growth Factor; D020574:Proto-Oncogene Proteins c-sis; D053773:Transforming Growth Factor beta1; D000077214:Becaplermin; D018995:Myosin Heavy Chains