Induction and Establishment Protocol of Induced Pluripotent Stem Cell Lines in Mice

• In 2006, Takahashi et al. screened 24 stem cell-related transcription factors and found that only four transcription factors, Oct4, Sox2, Klf4, and c-Myc, were required to reprogram mouse embryo fibroblast (MEF) into induced pluripotent stem cells (iPSCs). In 2007, Takahashi reprogrammed human somatic cells with the same four factors, and Yu obtained human iPSCs using Oct4, Sox2, Lin28, and Nanog; after that, the research and interest in iPSCs exploded.
• iPSCs are similar to embryonic stem cells (ESCs) in many characteristics, but they are more readily available than ESCs and are ideal for studying the mechanisms of targeted cell differentiation and differentiation.

• On Day 3 after transfection, i.e. Day 3, 1 12-well culture plate of feeder layer cells is prepared.
• On Day 4 after transfection, the transfected cells are digested with 0.05% trypsin, terminated with cell basal culture medium, and the cell suspension is collected into a 15 mL centrifuge tube and centrifuged at 1000 r/min for 5 min at room temperature. The supernatant is discarded, and the cells are resuspended with 5 mL of iPSCs induction medium and counted with a cell counting plate. The transfected cells are inoculated onto feeder cells in 12-well culture plates at a density of 1.25×10⁴ cells/well and cultured with an iPSCs induction medium, and the cells are changed daily.
• The changes in cell morphology are observed daily, and when clones with morphology close to that of normal mESCs appeared, the clones could be picked for monoclonal culture, generally 12-21 days after transfection. miPSCs cells had large nuclei, mostly euchromatin in the nucleus, little cytoplasmic cytoplasm, simple structure, closely arranged cells, and colony-like growth.
• Clone picking the day before, prepare multiple 4-well culture plates of feeder layer cells.
• 4 drops of 0.25% trypsin are prepared on the lids of multiple domestic 35 mm culture dishes and placed in the incubator.
• Pull several glass needles over an alcohol lamp, requiring a fine tip (the pore size should be equivalent to the size of a clone).
• Change the feeder layer cells of the 4-well culture plate for the miPSCs culture medium.
• Under the body microscope, pick the better clones with glass needles and transfer them to 0.25% trypsin drops for digestion for 5 min. Aspirate the finished clones into the 4-well culture plate and blow them repeatedly with a pipette gun until the clones are blown into single cells.

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• Induced pluripotent stem cells (also known as iPS cells or iPSC) are a type of pluripotent stem cell that can be generated directly from adult cells. We offer Mouse iPSC Line, which was derived from mouse embryonic fibroblasts (MEFs) by retroviral expression of Oct3/4, Sox2, Klf4, and c-Myc genes.

• The density of cells inoculated on feeder layer cells has a great impact on the induction efficiency. If the density is too high, significant cell death occurs in the late induction phase.
• Observations of cell status need to be performed daily.

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