Method for transforming stem cells from Primed state to Naive state
A stem cell, cell technology, applied in the field of cell biology, can solve problems such as insecurity
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Embodiment 1
[0039] Example 1 Transformation of primed state stem cells to naive state stem cells
[0040]First, spread 30× diluted Matrigel glue or feeder cells on the culture dish one day in advance. The next day, primed stem cells were seeded on 30× diluted Matrigel gel or feeder cells at a passage ratio of 1:6. Cells were inoculated in clusters and cultured in mTeSR1 medium for 48 hours. In order to convert the Primed state In the state of stem cells, the mTeSR1 medium was removed, and the transformation medium was added (recipe as shown in Table 1).
[0041] basal medium small chemical molecule F12 DMEM medium 0.1uM Dasatinib Neurobasal Medium 0.1uM AZD5438 1XB2 Supplement 0.1uM SB590885 1XN2 Supplement 1uM PD0325901 1XL-Glutamine 10uM Y-27632 1X-non-essential amino acids 20ng / mL IL6 / sIL-6R 0.1mM β-mercaptoethanol 20ng / mL Recombinant Human Activin-A 50ng / mL bovine serum albumin 8ng / mL basic fibroblast growth factor...
Embodiment 2
[0044] Example 2 Observing the morphology of stem cells in primed state and naive state
[0045] Human stem cells in Primed state: the clones are larger, flat, with sub-smooth edges. Humanity Stem cells: The clones are smaller, raised, and have smooth edges. like figure 1 , showing that the shape of Naive cells transformed by our transformation medium meets the requirements.
Embodiment 3
[0046] Example 3 Detecting the Expression of Stemness Genes in the Culture Process by qRT-PCR
[0047] The expression of stemness genes (KLF4, KLF5, DPPA3, DPPA5, DNMT3L, NANOG) was detected by qRT-PCR during culture.
[0048] First complete the RNA extraction. Use the RNA extraction kit RNeasy Mini Kit from QIAGEN, and operate according to the product instructions. The reagents and consumables used in the experiment are all RNase-free. The specific operation is as follows: A. CiPSCs remove feeder cells by differential attachment method, wash once with PBS, add 350 μl of Buffer RLT containing 1% β-mercaptoethanol after centrifugation, and resuspend fully by pipetting. B. Add 350 μl of 70% absolute ethanol, pipette and mix well, then transfer to the adsorption column, centrifuge at 10000g for 15 seconds, and discard the effluent. C. Add 350μl Buffer RW1, centrifuge at 10000g for 15 seconds, and discard the effluent. D. Add 10 μl DNaseI to 70 μl Buffer RDD, mix well, add ver...
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