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Application of TGF-beta inhibitor in inducing neural stem cells and organoid formation

A technology of neural stem cells and β inhibitors, applied in the application field of TGF-β small molecule inhibitors in the induction of neural stem cells and organoid formation, can solve the problems of undisclosed applications in the field of nerve regeneration, and achieve huge economic and social effects Effect

Pending Publication Date: 2021-11-23
IREGENE THERAPEUTICS LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the development direction of this molecule is mainly based on drug development and treatment of lung cancer, liver cancer and glioblastoma (Pharmaceutics.2020May18; 12(5):459), and does not disclose any application in the field of nerve regeneration

Method used

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  • Application of TGF-beta inhibitor in inducing neural stem cells and organoid formation
  • Application of TGF-beta inhibitor in inducing neural stem cells and organoid formation
  • Application of TGF-beta inhibitor in inducing neural stem cells and organoid formation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Example 1: Preparation of neural stem cell induced medium

[0055] The formulation of nerve induction base medium (hereinafter referred to as Nouvneu001): Du's improved Iguor / F12 medium (DMEM / F12), 1% minimum numerous non-essential amino acid (MINIMUM Essentialmedium Non-Essential Amino Acids , Thermo Fisher, Item No .: 11140076), Sodium Chloride (Sodiumchloride, 0.5 g / l), sodium selenate (SODIUM SELENITE, 13.6 μg / L), insulin (22 μg / ml), recombinant human transferrin (100 ng / ml).

[0056] The preparation of the neural stem cell inducing medium used in the present invention is to add 55 nm to 24 μm LY 2157299 (Selleck, S2230) to the NouvNeu001 base medium, and the final concentration is preferably 300 nm, 500 nm, 700 nm, 900 nm, 1 μm, 2.5 μm, 5 μm. 7.5 μm, 10 μm, 12.5 μm, 15 μm, 17.5 μm, 20 μm or 22 μm, most preferably 12.5 μm. The above medium referred to as a LY induced medium, and the experimental group using this medium is referred to as a Ly induction method...

Embodiment 2

[0058] Example 2: Inducing and identification of neural stem cells

[0059] 2.1 Chemical induction of neural stem cells:

[0060] Multi-capable cells include embryonic polycogene stem cells such as H9 cell lines and humans induced pluripotent stem cells. The human induced pluripotent stem cells used in the present invention are carried out in accordance with the culture method of "reprogramming medium and rejoinographic induced plurality of stem cells" (ZL201910050800.7 patent), from CD34 + Cell recombination is obtained.

[0061] Human multi-capable cells use Matrigel (STEMCELL TECHNOLOGIES) to incubate the T25 cell culture flask, and placed in a 37 ° C incubator for more than an hour or more. According to 1 × 10 6 The cells were inoculated in the T25 culture flask for amplification and passage.

[0062] When nerve-induced, 50 μg / ml polyethyl lysine (Sigma, Item No .: P6407) is used to incubate in a thermostat of 37 ° C for more than 3 hours, then use 5 μg / ml layer adhesion p...

Embodiment 3

[0077] Example 3: Differentiation function identification of neural stem cells

[0078] The neural stem cells obtained in the second Example 2 were directed in the NouvNeu001 base medium. When neuronal differentiation, 50 μg / ml of polylysine (SIGMA ALDRICH, ITR: P6407) was used to incubate in a thermostating tank in a 37 ° C incubation for 3 hours or more, until cellular inoculation.

[0079] 3.1 Pain Acceptor Neurons Differentiation:

[0080] The neural stem cells obtained in Example 2 were 1 × 10 5 The proportion of each bottle is inoculated in the T25 culture flask, and 3 μm Chir99021 (Selleck, Item No .: S2924), 10μm SU5402 (TOCRICK) (S2924), 10μm DAPT (S2924), 10 μm DAPT (SELLECK, IN: S2215), Fresh medium is replaced every 3 days until 21 days. Culture conditions were 37 ° C, 5% CO 2 . The experimental results show that the neural stem cells obtained in Example 2 have an axiscore structure, and the expression of the painful susceptor neuron marker SCN11A and Nestin ( Figure...

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Abstract

Provided is a new use of a TGF-beta small molecule inhibitor in the field of neuroregeneration, which can be used for the in vitro regeneration and directed differentiation of various nerve cells and brain-like organs. By adding the inhibitor to a set of basal media having clear chemical compositions, pluripotent stem cells can be induced into adult cells derived from a variety of neural stem cells, and the number of induced nerve cells and the size of organoids can be greatly increased. The induction system provided in the present invention expands new functions of a single small molecule in the field of ectodermal cell induction and differentiation and at the same time avoids the use of B27 and other serum substitutes, thereby completely avoiding the potential risks caused by the presence of animal-derived components in cell culture processes, and greatly expanding the clinical prospects of a variety of nerve cell transplantations.

Description

Technical field [0001] The present invention belongs to the biological field, and more particularly to the application of TGF-β small molecular inhibitors in inducing neural stem cells and category organ formations. Background technique [0002] The outer endoderm is the outermost layer formed in the embryonic development process. As the start of the organ, the formation cells are gradually divided into important systems such as brain, spinal cord, sensory organs. Among them, the nervous system is an important system responsible for thinking, emotion, perception, exercise and other functions. Compared to diseases such as tumors, the current number of drugs in the neurological disease is less, and the R & D cycle is long, and the most important reason is the particularity of various primary cells in the pendulum layer. For example, the primary neuron is not renewable. Sexual causes the scarce platform of nervous system drugs. [0003] In addition to drug screening, in vitro regene...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0797C12N5/0793C12Q1/02A61K35/30A61P25/00
CPCC12N5/0619C12N2506/45C12N2500/90C12N5/0623G01N33/5058A61K35/30A61P25/00C12N2500/25C12N2500/32C12N2500/12C12N2506/08C12N2533/52C12N2503/02G01N2500/10C12N2501/405C12N2501/15G01N33/5073G01N33/6896G01N2333/495Y02A50/30C12N5/062C12N2500/98G01N33/5088A61P25/28C12N2506/02C12N2533/54C12N5/0037C12N2500/24C12N2501/13C12N2506/03A61K31/4709C12N5/0696
Inventor 魏君蔡萌周佳侯梦莹云轩
Owner IREGENE THERAPEUTICS LTD
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