[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).

CONCLUSION: The human ADSCs can be induced to express vascular smooth muscle markers, and they are a new potential source of vascular tissue engineering.