A method for reprogramming human mononuclear cells into induced pluripotent stem cells
A technology of pluripotent stem cells and nuclear cells, which is applied in the field of reprogramming human mononuclear cells into induced pluripotent stem cells, can solve the problems of needing to prepare and use now, not, Vitronectin Matrigel is expensive, etc., and achieve long storage time and application The effect of good prospects and high induction rate
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Embodiment 1
[0038] Example 1 Preparation of human amniotic mesenchymal stem cell matrigel of the present invention
[0039] 1. Test method
[0040] 1.1 Aseptically obtain human amniotic membrane tissue, and then wash it with PBS for 3 times. Mechanically cut the tissue to about 0.2cm in aseptic operation 2 Size, use the tissue block attachment method to attach the tissue block to the Cellbind cell culture plate, add TheraPEAK containing 1% double antibody TM MSCGM-CD TM Medium, placed at 37 °C 5% CO 2 cultured in a cell culture incubator. When the confluence of the cells was greater than 90%, they were digested with 0.05% trypsin and passaged at a ratio of 1:4.
[0041] 1.2 When subcultured human amniotic mesenchymal stem cells (AM-MSCs) from P3 to P10 ( figure 1 A) When growing to more than 90% confluence, wash once with PBS, and lyse with 1% RIPA lysate, 5% RIPA lysate and 10% RIPA lysate at room temperature for 5-6 minutes respectively. Then wash with PBS 3 times, add 80% methan...
Embodiment 2
[0047] Example 2 Using Matrigel of the present invention to induce reprogramming of human mononuclear cells into human induced pluripotent stem cells
[0048] (1) Isolation and culture of human mononuclear cells
[0049] 1. Test method
[0050] 1.1 Transfer the umbilical cord blood (0.5-2ml) or peripheral blood (5-10ml) in the umbilical cord blood collection bag or anticoagulant tube to a sterile centrifuge tube, add an equal volume of PBS to dilute and mix well.
[0051] 1.2 Take a new centrifuge tube, add Ficoll separation solution, slowly transfer an equal volume of diluted blood to the surface of the Ficoll layer, and centrifuge at 600g for 20min at room temperature.
[0052] 1.3 After centrifugation, suck out the buffy coat cells, add PBS to wash once, and centrifuge at 600g for 5min.
[0053] 1.4 Resuspend cells with fully humanized erythroid medium (basal medium is H3000 and ACF medium from Stemcell Company respectively, and then add cytokines SCF 100ng / ml, IL-3 10ng / ...
Embodiment 3
[0074] Example 3 Using the human amniotic mesenchymal stem cell matrigel of the present invention to culture the iPSCs obtained by the reprogramming of the present invention
[0075] 1. PCR method to detect the integration of exogenous genes.
[0076] experimental method:
[0077] The ips clones obtained from the reprogramming in Example 2 were continuously cultured on the human amniotic mesenchymal stem cell matrigel prepared in Example 1 until P5, P7, and P12, and the genomic DNA was extracted using Tiangen’s Genomic DNA Extraction Kit , The concentration was determined with a Nanodrop 2000 ultra-micro spectrophotometer from Thermo Company. With the plasmid backbone EBNA as the detection object and GAPDH as the housekeeping gene, the PCR method was used to detect the integration of exogenous genes into genomic DNA.
[0078] Experimental results:
[0079] Such as Figure 9 As shown, the plasmid backbone EBNA can be detected in the genomic DNA of iPSCs P5 and P7, but the e...
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