Method for producing derivational pluripotent stem cell with meninges cell and uses thereof
A technology of pluripotent stem cells and cell induction, which is applied in the field of producing induced pluripotent stem cells from meningeal cells and its application, can solve problems such as tumor occurrence, and achieve a good survival effect
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0054] Cell Preparation and Culture
[0055] The meninges of newborn C57BJ / L6 mice were isolated with 0.25% trypsin-EDTA (Gibco ) at 37°C for 8-10 minutes and then gently dissociated with a fire-polished pipette. The sample was left to stand for 5 minutes to precipitate tissue fragments, and the obtained cell suspension was divided into 3.5×10 5 Cells / ml were seeded in 100-mm dishes pre-coated with poly-D-lysine (100 μg / ml). The obtained cells were cultivated with high glucose DMEM medium (glucose of 4.5g / L, HyClone Company), containing 10% fetal calf serum (HyClone Company), 2mM sodium pyruvate (Gibco Company), 2mM L-glucose in the medium Aminoamide (Gibco), penicillin (100 U / ml) and streptomycin (100 μg / ml).
[0056] Embryonic stem cells (ES) and induced pluripotent stem cells (induced pluripotent stem cells, iPS cells) were cultured on MEFs (mouse embryonic fibroblasts) treated with mitomycin (10 μg / ml), and cultured at high In glucose DMEM (HyClone company), the high ...
Embodiment 2
[0059] DNA microarray
[0060] Total RNA was isolated from cells using the RNAeasy min kit (Qiagen, Valencia, CA) according to the manufacturer's instructions. Using 3 mg of total RNA as a template, cDNA was synthesized using T7-Oligo (dT) promoter (Affymetrix, Santa Clara, CA) and Superscript II polymerase (Ivitrogen). An in vitro transcription system (using the RNA Transcript Labeling Kit (Affymetrix)) was used to synthesize biotin-labeled cRNA (according to the instructions of the Affymetrix RNA Transcript Labeling Kit).
[0061] Fragmented biotin-cRNA was hybridized to mouse GeneChip430.2 (Affymetrix). Use GeneArray Scanner 7G (Affymetrix) to scan the chip, according to the manufacturer's instructions, use the default parameters to process the data (CEL file form) with the Robust Multi-chip Average (RMA) method, and subtract the background from it (based on the default internal reference probe) , and normalized using the software ArrayAssist5.5.1 (Stratagene Corp. and St...
Embodiment 3
[0069] Viral vectors infect meningeal cells
[0070] According to the method described in Example 1, in the p6 well culture plate, about 4 × 10 per well 4 The meningeal cells of mice were inoculated with these cells, cultured overnight at 37°C and 5% CO2 under normal culture conditions, and the cultured meningeal cells were infected with the collected virus supernatant. The viral supernatant was obtained by transfecting Plat-E cells (Lipofectamine 2000, Invitrogen Company) with a retroviral pMX vector (Addgene Company) comprising mouse Oct4, Sox2, Klf4 and c-Myc cDNA according to conventional methods (See Sambrook, Fritsch and Maniatis, A Laboratory Guide to Molecular Cloning, 3rd Edition (2002)). Control incubations with vectors encoding GFP indicated transfection efficiencies approaching 100% (data not shown).
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More