Method for culture of urine-derived pluripotent stem cells by virtue of in vitro small molecule induction
A pluripotent stem cell and small molecule technology, applied in artificially induced pluripotent cells, non-embryonic pluripotent stem cells, animal cells, etc., can solve the problem of scarcity of stem cells, and achieve broad clinical application prospects, broad application prospects, enhanced The effect of blood flow restoration
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Embodiment 1
[0039] Example 1 Induction and culture of urine-derived pluripotent stem cells (hUSCs) by small molecules in vitro
[0040] The schematic diagram of the experimental steps for isolating and culturing hUSCs in vitro is as follows: figure 1 shown.
[0041] (1) Separation of urine-derived cells
[0042] Centrifuge 50-100mL of the obtained human urine sample at a speed of 500g for 5min, wash the pellet twice with PBS (phosphate buffered saline, pH7.2-7.4), discard the supernatant, and resuspend the cells with culture medium. It is: keratinocyte serum-free medium (KSFM) medium + 15% FBS (purchased from Gibco-Invitrogen Company), and the urine-derived cells are isolated;
[0043] (2) Primary culture of urine-derived cells
[0044] The obtained urine-derived cells were inoculated in a 24-well plate containing culture medium at a density of 500 cells / well, and the culture medium composition was: keratinocyte serum-free medium (KSFM) medium+10% FBS+penicillin (100U / ml )+Streptomyci...
Embodiment 2
[0049] Example 2 Small Molecule Induction and Culture of Urine-derived Pluripotent Stem Cells (hUSCs) in Vitro
[0050] A two-step induction method was adopted. In the first step, 5-aza-2'-deoxycytidine (5-AZA) was added to make the final concentration 2 μmol / L, and induced for 2 days; in the second step, 3- Deazaneplanocin A (DZNeP) and vitamin C (vitamin C) were induced, and the final concentrations reached 3 μmol / L and 40 μg / ml, respectively,
[0051] All the other steps are the same as in Example 1.
Embodiment 3
[0052] Example 3 Small Molecule Induction and Culture of Urine-derived Pluripotent Stem Cells (hUSCs) in Vitro
[0053] A two-step induction method was adopted. In the first step, 5-aza-2'-deoxycytidine (5-AZA) was added to make the final concentration 8 μmol / L, and induced for 2 days; in the second step, 3- Deazaneplanocin A (DZNeP) and vitamin C (vitamin C) were induced, and the final concentrations reached 4 μmol / L and 10 μg / ml, respectively,
[0054] All the other steps are the same as in Example 1.
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