A method for obtaining monkey haploid neural stem cells

A neural stem cell, haploid technology, applied in the field of bioengineering

Active Publication Date: 2021-04-09
NANKAI UNIV
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Problems solved by technology

[0003] To sum up, the problem existing in the prior art is that there is a technical gap in obtaining monkey haploid neural stem cell data through in vitro differentiation.

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  • A method for obtaining monkey haploid neural stem cells

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Embodiment 1

[0026] 1. Preparation of a pluripotent monkey embryonic stem cell culture medium

[0027] After improvement, the culture medium for the growth of haploid monkey stem cells contains D / F12 medium with 20% KOSR and 0.1mM non-essential amino acids, and other supplements in the medium are 0.1mM β-mercaptoethanol, 100U / ml penicillin, 0.1 mg / ml streptomycin, 2mM L-Glutamine, 5ng / ml fibroblast growth factor (bFGF), 10ng / ml human leukemia inhibitory factor (LIF), 0.5μM PD0325901, 3μM CHIR99021, 10μM SB203580 and 10 μM of SP600125.

[0028] 2. Obtaining haploid neural stem cells from rhesus monkeys

[0029] The macaque haploid embryonic stem cells in culture were digested into single cells with 0.05% trypsin, resuspended with EB culture medium and cultured in a suspension culture dish. On the first day of differentiation, 10 μM Y-27632 was added to the EB culture medium to reduce cell apoptosis. On the 5th day of differentiation, the EB culture medium was replaced with neural inducti...

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Abstract

The invention belongs to the technical field of bioengineering, and discloses a method for obtaining monkey haploid neural stem cells. The haploid embryonic stem cells are treated with 0.05% trypsin / EDTA for optimized cultivation on the basis of the original ordinary monkey stem cell culture medium. Cloned into a single-cell state, cultured in suspension with EB medium to form embryoid body spheres; from the first day of differentiation, the medium should be added with ROCK inhibitor Y-27632 to inhibit cell apoptosis; on the fifth day of EB The culture medium was changed to neural induction medium; the EB balls that initially formed the neuroectoderm were planted in a petri dish with substrate gel, and neural induction medium was added for differentiation; neural rosettes were picked and planted in 30 μg / ml polyornithine and 3 μg / ml laminin in the culture dish; after 14 days of culture, flow cytometry was performed to sort haploid cells. The present invention realizes for the first time that monkey haploid neural stem cells are obtained in vitro, and have the ability to differentiate toward the downstream direction of nerves.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, in particular to a method for obtaining monkey haploid neural stem cells. Background technique [0002] Because monkey haploid embryonic stem cell lines are difficult to establish (only one research group has established the cell line reported in the literature). Monkey haploid embryonic stem cells have weak differentiation ability, and there will be serious diploidization in the process of haploid neural differentiation. So there is no report to prove that monkey haploid neural stem cells can be obtained by in vitro differentiation. [0003] To sum up, the problem existing in the prior art is that there is a technical gap in obtaining monkey haploid neural stem cell data through in vitro differentiation. Contents of the invention [0004] Aiming at the problems existing in the prior art, the present invention provides a method for obtaining monkey haploid neural stem cells. Since the...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/0797
CPCC12N5/0623C12N2500/32C12N2500/44C12N2501/11C12N2501/115C12N2501/235C12N2501/727C12N2533/32C12N2533/52
Inventor 帅领王海松张文豪
Owner NANKAI UNIV
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