A kind of motor neuron and its preparation method and application
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A motor neuron and motor nerve technology, applied in the field of stem cell biology, can solve the problems of low efficiency, long culture time of mature motor neurons, low purity, etc., and achieve high survival rate, large scientific research application potential, and high cell viability Effect
Active Publication Date: 2022-03-11
安徽中盛溯源生物科技有限公司
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[0007] In order to solve the problems of long culture time, low efficiency or low purity in inducing and obtaining mature motoneurons in the existing technology, the present invention discloses a method and application of fast, efficient and simple differentiation of pluripotent stem cells to obtain motoneurons
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Embodiment 1
[0096] This embodiment discloses a method for preparing motor neurons, comprising the following steps:
[0097] S1: Differentiation of pluripotent stem cells into neuroepithelial cells;
[0098] S2: Neuroepithelial cells differentiate into motor nerve precursor cells;
[0099] S3: Motor neuron precursor cells differentiate into post-mitotic motor neurons;
[0100] S4: Post-mitotic motor neurons differentiate to mature motor neurons, that is, mature motor neurons are obtained.
[0101] Specifically, the steps are as follows:
[0102] (1) Culture of pluripotent stem cells (D-3-D0)
[0103] The pluripotent stem cells used in the experiment have undergone strict pluripotency verification (expressing various pluripotency markers, and can form teratomas including inner, middle and outer germ layers in immunodeficient mice). Pluripotent stem cells are normally cultured in their maintenance medium, the medium used is E8 or TeSR or other similar medium. In this example, human plur...
Embodiment 2
[0129] In the method of Example 1, on the 9th day of culture, the motor nerve precursor cells were frozen with the motor nerve precursor cell cryopreservation solution. The specific steps are:
[0130] Aspirate the medium, wash once with 1x DPBS (w / o Ca2+ / Mg2+), add TrypLE digestion solution, place at 37°C, 5% CO 2 Concentration, incubate in an incubator with saturated humidity for 8-12 minutes, shake gently to make the cells completely detach from the bottom of the culture dish. Transfer the cell suspension into a 1.5mL centrifuge tube, centrifuge in a handheld centrifuge for 10-15 seconds, discard the supernatant, add 1mL motor nerve precursor cell cryopreservation solution, and gently pipette 1-2 times to disperse the cells into single cells as much as possible And count, the number of frozen cells is recommended to be 2-3×106cells / tube, and stored in liquid nitrogen.
[0131] After cryopreservation of motor nerve precursor cells, the cell recovery rate reached more than ...
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Abstract
The invention relates to the field of stem cell biology, in particular to a motor neuron and its preparation method and application. The motoneuron expresses OLIG2+, MAP2+ and CHAT+, and also expresses one or any combination of PAX6+, HB9+, ISL1+, Synaptophysin+ and Tubulin+. The invention also discloses a method for preparing motor neurons, comprising the following steps: differentiation of pluripotent stem cells to neuroepithelial cells; differentiation of neuroepithelial cells to motor nerve precursor cells; differentiation of motor nerve precursor cells to post-mitotic motor neurons; mitosis After the motor neurons differentiate into mature motor neurons, mature motor neurons are obtained. The preparation method provided by the invention does not use a system containing serum or a serum substitute; the differentiation efficiency is high and the differentiation effect is stable; the purification method is simple and the obtained cells have high purity. The cryopreservation solution of motor neuron precursor cells is used, which has clear components and low price; the cell viability is high (>80%) after recovery, and the ability to efficiently differentiate into mature motor neurons is still maintained.
Description
technical field [0001] The invention relates to the field of stem cell biology, in particular to a motor neuron and its preparation method and application. Background technique [0002] Motor neuron disease (MND) is a group of chronic degenerative diseases that selectively invade the anterior horn cells of the spinal cord, brainstem motor neurons, cortical vertebral cells and vertebral tracts with unknown etiology. It is progressive degeneration, necrosis and apoptosis of upper and lower motor neurons. Due to the different combinations of symptoms and signs, different types of motor neuron diseases are formed, including amyotrophic lateral sclerosis (ALS), spinal muscular atrophy (SMA), primary lateral sclerosis ( primary lateral sclerosis (PLS) and progressive bulbar palsy (progressive bulbar palsy, PBP), among which ALS is the most common type of chronic motor neuron disease, commonly known as "gradual freezing syndrome". ALS has two forms: one is sporadic ALS (sALS), wh...
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