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Method for differentiating human induced pluripotent stem cells into oligodendrocytes, kit and application
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A technology of small molecule compounds and inhibitors, applied in the field of differentiation of human induced pluripotent stem cells to oligodendrocytes, can solve the problems of low efficiency and long time
Active Publication Date: 2021-10-29
CHENGNUO REGENERATIVE MEDICINE TECH (ZHUHAI HENGQIN NEW AREA) CO LTD
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iPSCs can also be directly induced into OPCs, but current techniques are time-consuming and inefficient
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Embodiment 1
[0120] Example 1. Inducing iPSC cell differentiation and verifying the induction effect
[0121] Prepare the following media for use:
[0122] Complete medium for neural induction: 98% DMEM / F-12 medium, 1% non-essential amino acids, 1% GlutaMAX-I, 0.1 mM 2-Mercaptoethanol, 10 μM SB431542, 0.25 μM LDN193189 and 100 μM tretinoin, 25 μg / ml insulin .
[0123] N2 medium: 97% DMEM / F-12 medium, 1% non-essential amino acid, 1% GlutaMAX-I, 0.1 mM 2-Mercaptoethano, 1% N2 supplement, 1 μM SAG and 100 μM tretinoin.
[0124] B27 medium: 95% DMEM / F-12 medium, 1% non-essential amino acid, 1% GlutaMAX-I, 0.1mM 2-Mercaptoethanol, 1% N2 supplement, 2% B27supplement and 1μM SAG and 100μM vitamin A acid, 25μg / ml insulin.
[0125] OPC maturation medium - without puerarin: 95% DMEM / F-12 medium, 1% non-essential amino acids, 1% GlutaMAX-I, 0.1mM 2-Mercaptoethanol, 1% N2 supplement, 2% B27supplement, 10ng / mL PDGF - AA, 10 ng / mL IGF-1, 5 ng / mL HGF, 10 ng / mL NT3, 60 ng / mL T3, 100 ng / mL Biotin, 1...
Embodiment 2
[0151] Embodiment 2, the effect of puerarin in OPC maturation medium on inducing oligodendrocytes
[0152] According to the method of Example 1, complete nerve induction medium, N2 medium, B27 medium, OPC maturation medium are configured; and OPC maturation medium not containing puerarin is configured; OPC maturation containing puerarin or not containing puerarin The medium was used as a control to explore the effect of puerarin on the induction of oligodendrocyte formation.
[0153] On the 37th day of culture, the percentage of oligodendrocytes in all cells was quantitatively counted, and the statistics were as follows: image 3 Immunofluorescence analysis was performed as indicated, and the results were as follows Figure 4 shown.
[0154] The OPC maturation medium without puerarin was used as the control group, and about 21% oligodendrocytes could be generated in the control group, and about 36% oligodendrocytes could be generated after adding puerarin. It proved that ...
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Abstract
The invention relates to the technical field of biology, in particular to a method for differentiating human induced pluripotent stem cells into oligodendrocytes, a kit and application. The method comprises the step of culturing stem cells by using at least one of the following culture media of a nerve induction complete culture medium, an N2 culture medium, a B27 culture medium and an OPC mature culture medium, and more preferably, the number of the oligodendrocytes induced by using the puerarin-containing OPC mature culture medium can be increased by 30% compared with the number of the oligodendrocytes induced by not using puerarin.
Description
technical field [0001] The invention relates to the field of biotechnology, in particular to a method, kit and application for human induced pluripotent stem cells to differentiate into oligodendrocytes. Background technique [0002] Oligodendrocyteprogenitor cells (OPCs) are central nervous system cells present in vertebrates, which form myelin sheaths to wrap around nerve fibers, and play a key role in the transmission of nerve signals and the nutrition and protection of nerve fibers . Oligodendrocytes produce a lipid-rich lamellar myelin sheath that matures to form myelin, which coats neuronal axons and creates defined electrically insulating segments to maximize action potential conduction velocity. Myelin is also important for axonal integrity and survival, and it has been shown that even small changes affecting oligodendrocytemetabolism can lead to neurodegeneration. The process of myelination is particularly important in humans because the human brain has a high c...
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