Inducing Mouse Embryonic Stem Cellto Vascular Smooth Muscle Cell In Vitro

Objective: To explore an optional condition of inducing mouse embryonic stem cells to differentiate into vascular smooth muscle cells(VSMC), and provide seed cells for vascular tissue engineering.Method: This experiment was finished in the Blood Disease Institute, the Second Affiliated Hospital of Nanchang University from August 2006 to February 2007.①Animal and cell line: One depuratory grade Kunming Mus musculus albus pregnant for 12.5d. The disposition of the animal was accord with the ethical standard.②Empirical method: Mouse embryo fibroblast (MEF) was segregated from the 12.5d fetus mouse. Condition medium: As the MEF was serial passaged to 3-5 generations, the Low DMEM was replaced by the High DMEM with 15% fetal bovine serum (FBS), the High DMEM was collected after 24h, and added 2.0mmol/L L- Gln-glutamine, 1×non-essential amino acid, 0.1mmol/Lβ-mercaptoethanol, 1000U/mL leukaemia inhibitory factor. ES cells were cultured in the T25 culture flask treated by 0.1% gelatin with the condition medium, and formed into embryoid body ( EB )differentiation model. The experiment was set up to 3 groups, in the experimental group 10-9mol/L all-trans-retinoic acid was added from the 3rd d to the 10thd, 3ng/ml TGF-β1 was added from the 7thd to the 10thd, and 20ng/ml platelet-derived growth factor-BB was used in this experiment from the 11thd to the 28thd. In the serum Control Group nothing inductor was added, and in the all-trans-retinoic group 10-9mol/L all-trans-retinoic was added. The EBs were digested into single cells by trypsinase and collagenaseⅡ,then were cultivated for 7d.③Experiment evaluation: RT-PCR was used to detect the expression of smooth muscleαactin (SMαA), and smooth muscle-myosin heavy chain (SMMHC). Immunofluorescence was used to analyze the express of the SMαA of the differentiated cells.Result:①ES cells could form embryoid body spontaneously for about 6 days, at the10th day many"round"cells appeared around embryoid body, and many fusiform cells appeared at the 14th day.②These fusiform cells could express SMαA and SM-MHC at the 21st day.③Immunocytochemistry: These VSMC-like cells were observed by fluorescence microscope. Cytolymph could emit red fluorescence with Rhodamine,and cellular nucleus could emit blue fluorescence with DAPI. The positive rate of cells in the experimental group was obviously higher than the control groups (P<0.05).Conclusion:①The culture system of feeder free with LIF could be used to probote the growth and proliferation of ES cells,maintain their undifferentiation and pluripotentiality. It will benefit to maintain their undifferentiation application of ES cells in next study.②ES cells could differentiate into vascular smooth muscle cells in some definite condition. With the solving of these questions, ESCs could provide seed cells for blood vessel tissue engineering and other vascular prostheses.