Method for preparing pig neural stem cells by reprogramming
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A technology of neural stem cells and cells, applied in the field of preparation of pig neural stem cells by reprogramming
Active Publication Date: 2014-04-23
INSITUTE OF BIOPHYSICS CHINESE ACADEMY OF SCIENCES
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However, unlike the reprogramming of other species, the exogenous reprogramming factors introduced during the reprogramming of pig somatic cells cannot be silenced, so the obtained cells are non-classical induction of continuous expression of exogenous stemness genes and e
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Embodiment 1
[0125] Example 1. Using reprogramming to obtain nerve cells from porcine embryonic fibroblasts
[0126] 1. Co-import
[0127] Pig embryonic fibroblasts (recorded in the following literature: Liu K., et al.Generation of porcine-induced pluripotent stem cells by using OCT4and KlF4porcine factors. Cell Reprogram.2012,14(6):505-513, public available from obtained from the Institute of Biophysics, Chinese Academy of Sciences) and cultured in vitro in porcine somatic cell culture medium.
[0128] First discard the original medium of porcine embryonic fibroblasts, then add TrypLE TM Express (Life Technology, USA, Cat. No.: 12605010) digested the cells at 37 degrees Celsius. After 5 minutes, the pig somatic cell culture medium was used to terminate the digestion reaction, and the cells were collected by centrifugation at 1000 rpm for 10 minutes, which were the pig embryonic fibroblasts to be introduced.
[0129] The Episomal plasmid vector pCXLE-hOCT3 / 4 expressing gene OCT3 / 4 (the n...
Embodiment 2
[0149] Example 2. Directed differentiation of porcine neural stem cells into functional nerve cells
[0150] 1. Obtaining functional nerve cells
[0151] The porcine neural stem cells prepared in Example 1 were inoculated on a culture plate coated with Magrigel (purchased from BD Biosciences, USA, product number: 354277), and neural differentiation medium was added for continuous culture for 3 weeks to obtain functional porcine neural cells.
[0152] 2. Detection of functional nerve cells
[0153] The cell morphology of the porcine functional nerve cells obtained in the above 1 is detected, and the morphology of the generated nerve cells is detected by indirect immunofluorescence whether they are neurons or glial cells.
[0154] The pig functional nerve cells obtained in the above 1 were subjected to indirect immunofluorescence staining, and the results were as follows: image 3 As shown in A, it can be seen that the porcine induced neural stem cells have neural differentiat...
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Abstract
The invention discloses a method for preparing pig neural stem cells by reprogramming. The invention provides a method for preparing mammal neural stem cells by reprogramming. The method comprises the following steps: (1) commonly conducting gene OCT3/4, SOX2, KLF4 (Kruppel-Like Factor 4), LMyc and LIN28 into mammal somatic cells by using a non-integrated type episomal carrier to obtain transgenic cells; and (2) culturing the transgenic cells by using an induction culture system containing sodium butyrate to obtain the neural stem cells. An experiment shows that the non-integrated type episomal carrier is used for conducting factors including the OCT3/4, the SOX2, the KLF4, the LMyc, the LIN28 and the like into pig somatic cells; the somatic cells are transdifferentiated into the neural stem cells through optimizing a cultivation condition; the neural stem cells are further differentiated to obtain neural cells with physiological activity so as to lay foundation to nervous system diseases taking a pig as a model.
Description
technical field [0001] The invention relates to the field of biotechnology, in particular to a method for preparing pig neural stem cells by reprogramming. Background technique [0002] Neural stem cells have broad prospects in the basic research of neurobiology and cell therapy of nervous system diseases. For a long time, the source of neural stem cells has always been limited to the fetal brain or the directed differentiation of pluripotent stem cells, which severely limits its further application. At present, a new type of transdifferentiation technology provides a new source of neural stem cells. Neural transdifferentiation technology is a technology that directly transforms adult terminally differentiated cells into neural cells. This technology can quickly and directly obtain nerve cell materials without going through the pluripotent stem cell stage, so it will be widely used in nerve restorative therapy. At present, neural cell transdifferentiation has been success...
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IPC IPC(8): C12N15/85C12N5/10C12N5/0797C12N5/0793
Inventor 刘光慧曲静徐秀玲付丽娜杨济平任若通刘林刘凯
Owner INSITUTE OF BIOPHYSICS CHINESE ACADEMY OF SCIENCES
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