Preparation method of human-induced pluripotent stem cells
A cell and pluripotency technology, applied in stem cell engineering and biological fields, to achieve high reprogramming ability, growth-friendly proliferation, and non-invasive reverse differentiation effect.
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Embodiment 1
[0032] The preparation of embodiment 1 human deciduous tooth cells
[0033] Take natural deciduous or extracted human deciduous teeth, and separate dental pulp cells. The specific operation is as follows: draw a groove that reaches the pulp cavity at the junction of enamel and bone, split the tooth, take out the pulp tissue with small forceps and place it in Hanks solution, and at the same time rinse the pulp cavity with Hanks solution and merge it into the pulp tissue In the Hanks solution, the enzyme ophthalmic scissors was cut into pieces, and the final concentration of 2mg / mL neutral protease II and 0.5mg / mL type I collagenase were added to treat for 15min, repeated pipetting, and the Hanks solution containing 5% BSA was added to terminate the reaction. Dental pulp cells were obtained by sieving with metal mesh.
[0034] The deciduous tooth tissue from which the pulp was removed was crushed to an average particle size of 50-200 mesh, and treated with dispase I at a final ...
Embodiment 2
[0036] Example 2 Induced reprogramming of mammary tooth cells
[0037] The purchased mouse embryonic fibroblasts were used as 1 × 10 4 CFU / cm 2 Inoculate the cell culture flask at a concentration of 10% fetal bovine serum, add 1640 complete medium, place at 37°C, 5% CO 2 Cultivate in the incubator, change the liquid overnight, and observe with an inverted microscope until the cell confluency is about 70-75% for later use ( figure 1 ).
[0038]According to the method of Shinya Yamanaka, the expression cassettes of Oct4, Sox2, c-Myc and Klf4 factors were inserted into the retroviral vector, and the recombinant virus was co-transfected with the viral packaging vector into 293T cells, packaged to produce lentivirus, and the virus was collected solution for titer determination. Inoculate the human mammary tooth cells subcultured for 1-3 generations with the virus liquid at an MOI of 1:40, at 37°C, 5% CO 2 After incubating for 30 minutes, add 1640 complete medium containing 10%...
Embodiment 3
[0039] Example 3 Verification of IPS from human deciduous teeth
[0040] Subculture human deciduous teeth IPS vials, take a vial of subcultured human induced pluripotent stem cells, discard the previous complete human embryonic stem cell medium, wash twice with Hanks solution, add 4% paraformaldehyde (PFA) to fix 2 Minutes, after washing, add alkaline phosphatase staining solution, incubate for 15 minutes, wash with Hanks solution twice, and observe under an inverted microscope, the cell clone clusters are blue-purple and deeply stained, while the fibrous feeder layer cells around the cell clusters are not stained , indicating that the cells in the cell clone are in an undifferentiated state.
[0041] Take 1×10 5 CFU of IPS cells were injected into the medial thigh of NOD / SCID mice. After four weeks, tumor growth was observed. Eight weeks later, the tumor was removed, and the tissue sections were stained with HE and observed under a microscope. The results are shown in Figure...
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