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Method for screening pluripotent cell and special culture medium thereof
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A technology for screening pluripotent cells and culture media, which is applied in the field of screening pluripotent cells, and can solve problems such as lack of methods and inducible pluripotent cells not yet obtained
Inactive Publication Date: 2009-06-03
PEKING UNIV
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However, there is still no effective method for how to screen out induced pluripotent stem cells, and induced pluripotent cells with the same or similar morphology as mouse embryonic stem cells have not yet been obtained.
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Embodiment 1
[0068] Example 1, Screening of mESC-like human pluripotent stem cells
[0069] 1. Acquisition of human fibroblasts (target cells)
[0070] 1) The fresh embryonic foreskin (Fsf) was sterilized with 75% ethanol and washed with phosphate buffered saline (PBS) (fetal lung (FL) or adult foreskin (HFF) can be used instead of embryonic foreskin (Fsf)).
[0071] 2) Carefully separate the subcutaneous tissue with ophthalmic scissors and cut it into pieces.
[0072] 3) Wash several times with PBS, take small tissue pieces and inoculate them in culture dishes, and place them in a 37°C, 5% carbon dioxideincubator.
[0073] 4) After two hours, add DMEM high glucose medium II.
[0074] 5) After culturing for 2-3 days, remove the tissue pieces that cannot adhere to the wall.
[0075] 6) Continue culturing for 7-9 days, and remove the remaining tissue pieces.
[0147] Example 2, Screening of mESC-like Rat Pluripotent Stem Cells
[0148] 1. Acquisition of rat fibroblasts (target cells)
[0149] 1) Remove the head, limbs and viscera of fresh 12.5-day-old rat embryos, and wash them with PBS.
[0150] 2) Shred the tissue with ophthalmic scissors.
[0151] 3) Wash several times with PBS, take small tissue pieces and inoculate them in culture dishes, and place them in a 37°C, 5% carbon dioxideincubator.
[0152] 4) After two hours, add DMEM high glucose medium II.
[0153] 5) After culturing for 2-3 days, remove the tissue pieces that cannot adhere to the wall.
[0154] 6) Continue culturing for 7-9 days, and remove the remaining tissue pieces.
[0155] 7) Digest the cells with the digestion solution at room temperature for 5 minutes, neutralize them with DMEM high glucose medium II, and inoculate them in a new culture dish at a ratio of 1:3.
[0156] 8) After that, the medium was changed every 2 days, and the cells were subcultured ...
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Abstract
The invention discloses a method for screening a pluripotent cell and a special culture medium thereof. A pluripotent cell screening culture medium provided by the invention comprises PD0325901 shown in formula (I) and CHIR99021 shown in formula (II). The method for screening a pluripotent cell provided by the invention comprises the step of using the screening culture medium to culture a cell A, thus obtaining the pluripotent cell; and the cell A is a cell obtained by inducing an inducible factor encoding gene in a differentiation cell. The pluripotent cell obtained by applying the method of the invention has the protuberant cloned shape appearance of mESC; the symbol gene and the surface protein of an express embryonic stem cell have the activity of surface alkaline phosphatase and the capacity of automatically differentiating to form an Embryoid body as the embryonic stem cell;, and the pluripotent cell can be further differentiated into the cell types of an inner embryonic layer, a middle embryonic layer and an outer embryonic layer. The invention has important significance for the research field of the pluripotent cell.
Description
technical field [0001] The invention relates to a method for screening pluripotent cells and a special culture medium thereof. Background technique [0002] (1) Isolation of pluripotent cells [0003] In 1981, embryonic stem cells (mESC) were first isolated from mouse blastocysts (Evans et al. Nature 1981; 292: 154-156; Martin et al. Proc Natl Acad Sci USA 1981; 78: 7634-7638). In 2007, mouse epiblast stem cells (EpiSC) were successfully isolated (Brons et al. Nature 2007; 448: 191-195; Tesar et al. Nature 2007; 448: 196-199). The expression patterns of EpiSC and mESC are similar, but EpiSC and mESC are quite different in morphology and culture conditions, and they do not have the ability to embed into the germline after injection into recipient embryos. Recently, another new type of pluripotent cell FAB-ES was also established (Chou et al. Cell 2008; 135:449-461). FAB-ES also has a similar expression pattern to mESC and EpiSC. Although its morphology and culture conditio...
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