The His-tag fusion proteins were purified using Ni-NTA agarose

rse of early development, Schwann cells not only express lineage restricted differentiation markers such as nerve growth factor receptor, glial fibrillary acidic protein and S100b, but also up-regulate lipogenic gene expression. SREBP family transcription factors are the main regulators of lipogenic genes, which include the low density lipoprotein PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19674025 receptor and enzymes like HMG-CoA reductase and NAD dependent steroid dehydroge- 1 Early Schwann Cell Differentiation of Amniotic Fluid Stem Cells nase like . Recently, mTORC1 was suggested to be involved in SREBP activation and it was shown that conditional deletion of mTOR in mice resulted in a reduced myelin production by Schwann cells and reduced nerve conduction. The underlying mechanism, however, is still unclear. In the present study we investigated whether monoclonal human AFS cells can be used to generate early Schwann cells and analyzed the role of mTORC1 during this process. We applied a novel protocol to differentiate Schwann cells from AFS cells and demonstrated that inhibition of mTORC1 efficiently blocks Schwann cell differentiation, whereas induction of lipogenic genes stimulated Schwann cell differentiation. Transient transfection for gene overexpression The HA-S6K1-RR construct used in PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19673983 this study was purchased via Addgene: HA-S6K1-F5A-E389-R3A, rapamycin-resistant/153-18-4 web constitutively active. The differentiated AFS cells on day 15 were transfected with HA-S6K1-RR plasmid using Lipofectamine 2000 transfection reagent and cells were kept in differentiation media for another 72 hours. Immunofluorescence staining Cells cultured on chamber slides or 48-well plate were fixed in 4% paraformaldehyde at room temperature for 30 min. After fixation, cells were treated with PBS containing 0.1% Triton X-100 for 5 min at room temperature and then blocked with PBS containing 1% BSA for 30 min. Subsequently cells were incubated with primary antibodies diluted in PBS containing 1% BSA overnight at 4uC. The following antibodies were used: anti-NTRp75, anti-Glial Fibrillary Acidic Protein, anti-S100b, anti-nestin, anti-S6 ribosomal protein phosphorylated at S240/244, anti-LDLR, anti-HMGCR. Subsequently cells were washed and incubated with the secondary antibodies Alexa Fluor 546 goat anti-mouse IgG or Alexa Fluor 546 goat anti-rabbit IgG at room temperature for 1 hour. For visualizing nuclei, cells were stained with 6 diamidino-2-phenylindole dihydrochloride. The negative controls were generated by incubating with isotype specific control antibodies and omitting the first-step antibodies used in each experiment. Cells were observed using a fluorescence microscope. Quantification of immunofluorescence staining was performed by two independent researchers who were blinded regarding experimental details. A minimum of 250 cells per experiment were evaluated and cells with a staining intensity stronger than the isotype control stain were regarded as positive. Materials and Methods Cells, cell culture of human AFS cells The monoclonal human amniotic fluid stem cell line Q1 and a high Oct4 expressing single cell clone derived from the CD117/2 population was used in the study. Cells were maintained in a-MEM supplemented with 15% Fetal Bovine Serum, 18% Chang B, 2% Chang C, 2.5 mM L-Glutamine, 50 mg/L streptomycin sulphate and 30 mg/L penicillin. For neural crest marker expression melanoma-derived MCM1 cells were used as positive control. All cells were cultivated at 37uC in 5% CO2. Differentiation of human A