Reprogramming method for efficient inducing of T cells into multipotent stem cells
A technology for pluripotent stem cells and reprogramming, applied in animal cells, vertebrate cells, cell culture active agents, etc., can solve the problem of iPSC preparation technology failing to achieve large-scale preparation, and achieve the effect of high immune cell differentiation efficiency
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[0070] Example 1: Design and construction of episomal vector expression system
[0071] Such as figure 2 As shown, in this example, three episomal vectors were constructed, all of which were amplified by direct polymerase chain reaction (PCR) from some or all open reading frames (ORF) (using coding regions) from pluripotency determining factor genes. The first and last 20-22 bases are used as primers), and the above ORF is inserted into the commercially available mammalian expression vector pCEP4 or the related backbone containing OriP / EBNA1 to generate an episomal vector. All three episomal vectors contain at least An internal ribosome entry size (IRES), the first vector is pEP4-E-O2S-E-N2K( figure 2 A), which in turn contains the first promoter, POU5F1, IRES2, SOX2, the second promoter, NANOG, IRES2 and KLF4; the second vector is pEP4-E-O2S-E-T2K( figure 2 B), which in turn contains the third promoter, POU5F1, IRES2, SOX2, the fourth promoter, SV40LT, IRES2 and KLF4; the thir...
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[0072] Example 2: Primary isolation, culture and identification of T cell monocytes
[0073] 1. Primary isolation and culture of T cell monocytes
[0074] Collect a 6ml blood sample, transfer it to a lymphocyte separation tube, centrifuge, take the mononuclear cell layer, wash twice with DPBS, sample and count, and use flow cytometry to detect the expression of the surface molecules CD3+ and CD4+CD8+. Detect the positive rate and average fluorescence intensity indicators separately (the experimental results are as image 3 A), take 6×10 according to the counting result 6 Cells were seeded in 3 wells in a 6-well plate coated with activator, 2ml / well of T cell serum-free medium was added, and placed at 37°C, 5% CO 2 The culture in the incubator constitutes the expansion culture system of the present invention, and each well is supplemented with 1 to 2 ml of fresh T cell serum-free medium on the first day of expansion.
[0075] The serum-free medium for T cells in this embodiment includ...
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[0080] Example 3: Episomal vector induces reprogramming
[0081] 1. Method
[0082] a. Take 0.5~4×10 T cells activated and cultured for 2 days in Example 2 6 , Using the pEP4-E-O2S-E-N2K, pEP4-E-O2S-E-T2K and pCEP4-LM-2L episomal vectors constructed in Example 1 to electrotransfect target cells, and then inoculate them in hiPSC induction medium and Matrigel Or cultured in a six-well plate coated with vitronectin or other cell matrix, the transfection content of each plasmid DNA is pEP4-E-O2S-E-N2K: pEP4-E-O2S-E-T2K: pCEP4-LM -2L=1:1:1.
[0083] b. After 48 hours, replace the fresh hiPSC induction medium with half the amount, continue to culture for 10 days, and change the medium the next day, that is, reprogram on the feeder-free system.
[0084] The specific components of the pluripotent stem cell induction medium are:
[0085] One or more of the following small molecules were added per liter of the serum-free expansion medium for T cells in Example 2: CHIR99021 1 μmol, A-83-01 0.5 μ...
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