Schwann cells and method for preparing same
A cell and somatic cell technology, applied in the field of Schwann cells and their preparation, can solve the problem of direct conversion of no somatic cells into Schwann cells, and achieve the effect of avoiding cancer
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[0058] The invention relates to a preparation method of Schwann cells. The production method of the present invention is a method for producing Schwann cells without using pluripotent stem cells such as ES cells or iPS cells.
[0059] Schwann cells
[0060] Schwann cells are glial cells in the peripheral nervous system. Under physiological conditions, it supports nerve tissue, forms myelin (myelin sheath) and contributes to the realization of jumping conduction, etc. When the peripheral nerve is injured, it performs many important functions in the regeneration of the peripheral nerve, such as the production and release of neurotrophic factors, the basis of regenerated axons, and the formation of myelin.
[0061] Native Schwann cells originate from the neural crest, as opposed to neural cells derived directly from the ectoderm. Mature Schwann cells are formed through Schwann precursor cells, immature Schwann cells. In this specification, for simplicity, all cells in the m...
Embodiment 1
[0162] The outline of embodiment 1 method ( figure 1 )
[0163] figure 1 The outline of the method for producing the Schwann cell of the present invention ("directly reprogrammed Schwanncell:dSC" in the figure) is shown.
[0164] The cDNA coding sequences of various genes such as SOX10 were inserted into the retroviral vector plasmid pMXs.puro using the Gene art system. Suspend the packaging cell Plat GP cells in 1% NEAA 10% FBS DMEM containing 100 U / mL penicillin and 100 μg / ml streptomycin (common medium), and place them in gelatin-coated 10 cm culture dishes at a rate of 5× per plate. 10 6 concentration of individual sows (day -3). After 24 hours of culture, pMXs vectors containing various genes were simultaneously introduced in various combinations with pCMV VSV vectors using X-tremeGENE 9 at the following ratios. That is, a mixture of 5 µg of the introduced gene, 2.5 µg of pCMV.VSV, 500 µl of Opti-MEM, and 2.5 µl of X-tremeGENE 92.5 µl was added to a 10 cm dish filled...
Embodiment 2
[0165] Example 2 Conversion from human normal skin fibroblasts to Schwann cells, S100β fluorescent immunostaining image (Table 1)( figure 2 )
[0166] Human normal skin fibroblasts (aHDF) were cultured on a 12-well plate, and the operations described in Example 1 were performed. On the 14th day, the culture medium was removed from each well by suction, washed once with PBS, and then fixed with 4% PFA. Add blocking solution and let stand at 37 °C for 15 min. Cells stained with anti-S100β antibody (primary antibody) and AlexaFluor566-labeled anti-rabbit IgG antibody (secondary antibody). Different gene combinations are introduced into each well of the plate, and which gene combination is introduced into which number of wells is described in Table 1 (in the table, there is a material described as "1" in the column of each gene, indicating that the gene containing The retroviral vector of the gene is infected, and the blank column indicates that the retroviral vector conta...
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