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Midbrain dopaminergic nerve precursor cells and preparation method and application thereof

A neural precursor cell, dopaminergic technology, applied in the field of stem cell biology, can solve the problems of low efficiency, long differentiation method, unstable effect, etc., and achieve the effect of efficient differentiation

Inactive Publication Date: 2019-06-14
安徽中盛溯源生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] The invention relates to a midbrain dopaminergic neural precursor cell and its preparation method and application. The midbrain dopaminergic neural precursor cell has strong stability and can be expanded to 4-5 generations in large quantities. The disclosed preparation method can be fast and efficient Obtain midbrain dopamine cells from human pluripotent stem cells, which solves the problems of long time, unstable effect, low efficiency and the use of serum-containing culture system or trophoblast cells in the existing differentiation method, which is not conducive to subsequent clinical-grade cell preparations. Production and other issues

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  • Midbrain dopaminergic nerve precursor cells and preparation method and application thereof
  • Midbrain dopaminergic nerve precursor cells and preparation method and application thereof
  • Midbrain dopaminergic nerve precursor cells and preparation method and application thereof

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Experimental program
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Embodiment 1

[0064] This embodiment discloses a method for preparing midbrain dopaminergic neural precursor cells, such as figure 1 As shown, the specific steps are as follows:

[0065] D-1-D0: Formation of embryoid bodies (EBs)

[0066] First culture human pluripotent stem cells, digest undifferentiated human pluripotent stem cells in a good state and culture to a confluence of 70-90% into a single cell suspension, resuspend in human pluripotent stem cell maintenance medium at a certain density, Shake and culture overnight on a shaker in a 37°C incubator to form EBs with a relatively uniform size and shape.

[0067] Wherein, the above-mentioned human pluripotent stem cells are prepared by the method disclosed in patent CN 108085299A. It should be understood that the above-mentioned human pluripotent stem cells are not limited by source, and may include human pluripotent stem cells obtained by any means.

[0068] D-1-D0 experimental operation details and optimization are as follows:

[...

Embodiment 2

[0094] In this embodiment, the dopaminergic neural precursor cells obtained in Example 1 are identified, and the identification methods are as follows:

[0095] Method 1: Use RT-QPCR (real-time fluorescence quantitative PCR) to detect the indicators of forebrain, diencephalon, midbrain and hindbrain to determine the type of differentiated cells. The specific operation steps of RT-QPCR adopt conventional technical methods, and the specific experimental data are as attached Figure 4 As shown, among them, Figure 4 The middle ordinate refers to the expression level relative to hPSC, and the expression level of the target gene in hPSC is "1". From the figure, it can be concluded that the phenotype of the dopaminergic neural precursor cells identified in the experiment is: Lmx1a+ / Foxa2+ / En1+ / Otx2+ / Nkx2.1- / DBX1- / GBX2-.

[0096] Among them, Nkx2.1 is the indicator of the forebrain and diencephalon; DBX1 is the indicator of the front midbrain; Lmx1a, Foxa2, En1 and Otx2 are the ind...

Embodiment 3

[0101] Example 3 Optimization of CHIR99021 Concentration

[0102] The purpose of this example is to optimize the concentration of CHIR99021. Experiments have found that CHIR99021 treatment can significantly increase the ratio of Lmx1a+ / Foxa2+ / En1+ cells in EBs on day 11, and almost no expression of miscellaneous cells in other brain regions. The specific operation is as follows: When using T25 culture flasks for EB differentiation, other conditions and methods are as described in Example 1, and the concentration of CHIR99021 is gradient optimized, that is, 0 μM, 0.5 μM, 0.8 μM, 1 μM, 1.5 μM or 3μM CHIR99021 treated the cells, and used RT-QPCR to measure the specific indicators of each brain region on D11, the results are shown in the appendix Figure 7 . Figure 7 , the ordinate is the relative value of each group relative to the expression level of the target gene at CHIR=1 μM (the value of the expression level of the target gene at CHIR=1 μM is “1”).

[0103] It can be see...

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Abstract

The invention relates to the field of stem cell biology, in particular to midbrain dopaminergic neural precursor cells and a preparation method and application thereof. The midbrain dopaminergic neural precursor cell expresses Lmx1a+, Foxa2+, En1+ and Otx2+, wherein the midbrain dopaminergic neural precursor cell does not express one or more of Nkx2.1, DBX1 and GBX2. The method for preparing the mesencephalic dopaminergic neural precursor cell comprises the following steps: embryoid bodies are formed, midbrain floor cells are obtained, and midbrain dopaminergic neural precursor cells are obtained. By adopting the preparation method, the mesencephalic dopaminergic nerve cells can be rapidly and efficiently differentiated from the human pluripotent stem cells, and the problems are solved that an existing differentiation method is long in time, unstable in effect and low in efficiency, the culture system containing serum or the trophoblast cells are not conducive to the production of subsequent clinical-grade cell preparations and the like.

Description

technical field [0001] The invention relates to the field of stem cell biology, in particular to a midbrain dopaminergic neural precursor cell and its preparation method and application. Background technique [0002] Parkinson's disease is one of the most prevalent neurodegenerative diseases, mainly affecting middle-aged and elderly people. Symptoms include involuntary tremors of the hands, head, or mouth at rest, muscle stiffness, slowness of movement, and disturbance of postural balance. The etiology of Parkinson's disease is still unclear, but its main pathological feature is the selective loss of a large number of dopaminergic neurons in the substantia nigra of the midbrain, resulting in insufficient synthesis and secretion of dopamine. Dopamine is needed in the brain to direct muscle movement, and a lack of enough dopamine can affect motor skills, language skills, and other functions. The current treatment of Parkinson's disease mainly relies on levodopa preparations,...

Claims

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Application Information

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IPC IPC(8): C12N5/079A61K35/30A61P25/16
CPCC12N5/0618A61K35/30A61P25/16C12N2501/999C12N2506/45C12N2506/03
Inventor 焦璐琰俞君英张颖张东明许晗
Owner 安徽中盛溯源生物科技有限公司
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