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Direct de-differentiation of urine cell into nueral stem cell using synthetic messenger RNA

A neural stem cell and reprogramming technology, applied in the field of direct reprogramming of urine cells into neural stem cells using synthetic mRNA, can solve the problems of difficult clinical application, poor convenience, and low possibility, and achieve the goal of enhancing clinical feasibility Effect

Pending Publication Date: 2021-06-22
STEMLAB
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, currently performed direct reprogramming methods mainly use mouse cells as a cell source, so the possibility of regenerating in the same way in human somatic cells is not high
In addition, skin cells (fibroblasts) are frequently used among human somatic cells, but in this case, an invasive method of collecting the source of the cells is required, and this poses pain and safety risks to the donor, and convenience Sexual deterioration
In addition, clinical application is difficult because the method mainly uses viruses by introducing required reprogramming factors (genes) in the direct reprogramming process, and the gene integration system using viruses is not suitable for treatment because the system will be damaged due to selective insertion into the genome to induce mutation

Method used

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  • Direct de-differentiation of urine cell into nueral stem cell using synthetic messenger RNA
  • Direct de-differentiation of urine cell into nueral stem cell using synthetic messenger RNA
  • Direct de-differentiation of urine cell into nueral stem cell using synthetic messenger RNA

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] Example 1: Isolation of urine cells from urine

[0071] Based on a technique developed in England by Sutherland and Bain in 1972, details are given below. First, the donor-donated urine was centrifuged at 1000g for 10 minutes. After removing the supernatant, dilute the pellet remaining in the lower layer with 20 ml of PBS solution containing 1% penicillin / streptomycin / amphotericin B antibiotics. The diluted PBS+precipitation solution was then centrifuged at 1000g for 10 minutes. After removing the supernatant again, dilute with 1 ml basal medium (DMEMF12-based medium containing 1% penicillin / streptomycin / amphotericin B antibiotics, 1% L-glutamine, and 10% FBS) to leave the lower layer pellet and seeded onto gelatin-coated 12-well cell culture dishes. Next, after culturing the cells for 3 days by adding 1 ml of basal medium, the medium was replaced with growth medium (containing 1% penicillin / streptomycin antibiotics based on the medium obtained by mixing DMEM and REG...

Embodiment 2

[0072] Example 2: Introduction of reprogramming factors into urine cells using electroporation

[0073] The method of introducing the synthesized mRNA into the urine-derived cells cultured in Example 1 by electroporation is as follows. Synthesized mRNA was synthesized by a typical in vitro transcription kit (RiboMAX ◎ large-scale RNA production system, Promega), and the DNA to be the backbone of the mRNA to be synthesized was T7-VEE-OKS-iG (Steven Dowdy, Addgene), T7 -VEE-OKS-iG includes the genes of OCT4, KLF4, SOX2 and GLIS1 ( figure 1 ). As the sequences of OCT4, KLF4, SOX2 and GLIS1 genes, use those sequences disclosed in Nature.2011Jun 8; 474(7350):225-9, and for the corresponding gene sequence and mRNA application method, refer to Cell Stem Cell.2013Aug1 ; 13(2):246-54 and U.S. Patent No. 9,862,930B2.

[0074] By electroporating 3 times at 1600V, 10ms for 1 hour, 1×10 cells pretreated with 0.2ug / ml B18R protein were treated with 0.5μg synthetic mRNA. 6 Urine-derived ...

Embodiment 3

[0076] Example 3: Induction of Urine-Derived Reprogrammed Neural Stem Cells

[0077] After inoculating the urine-derived cells introduced with mRNA onto Matrigel-coated TM After being placed in a cell culture dish, the cells were cultured for 7 to 10 days in neural stem cell induction medium based on neural stem cell induction medium (by mixing DMEM / F12 medium and neural basal (Gibco) medium by 1: 1 Mix and add 1×N2 (Gibco), 1×B27 (Gibco), 10ng / ml human LIF, 2uM SB431542 (TGF-β inhibitor) and 3uM CHIR99021 (GSK3-β inhibitor) to the medium mixture medium) supplemented with 0.5uM purmorphamine (Shh agonist), 10uM Forskolin (adenylate cyclase activator), 100uM sodium butyrate (histone deacetylase inhibitor) and 64ug / ml ascorbyl 2-phosphate. When induction is complete, the development of neural stem cell colonies can be confirmed ( image 3 ).

[0078] In addition, Shh agonists, adenylyl cyclase activators, histone When one or more of acetylase inhibitors and ascorbic acid 2-p...

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Abstract

The present invention relates to a method for inducing a de-differentiation of neural stem cells from urine cells by introducing mRNAs of de-differentiation factors Oct4, Sox2, Klf4, and Glis1; and a composition for the prevention or treatment of neurological damage diseases comprising the neural stem cells induced by the method as an active ingredient.

Description

technical field [0001] The present invention relates to a method for directly reprogramming urine cells into neural stem cells and a pharmaceutical composition for treating nerve injury diseases, the pharmaceutical composition comprising the reprogrammed neural stem cells prepared by the method. Background technique [0002] Stem cells are cells that have unlimited potential for self-renewal or differentiation in relation to specific cells and tissues desired in the body. Stem cells are divided into three types: embryonic stem cells (ES cells) isolated from early embryos, embryonic germ cells (EG cells) isolated from embryonic primordial germ cells, and multipotent adult progenitor cells (MAPC cells) isolated from adult tissues. [0003] Since stem cells have the potential to differentiate into cells with specific functions, stem cells have been the subject of research as a cell therapy agent for restoring the functions of various organs, and recently, the application of ste...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0797C12N5/10A61K35/30A61P25/28A61P25/16A61P25/14A61P25/08A61P25/00A61P9/10
CPCA61K35/30A61P25/28C12N5/0623C12N2506/25C12N2533/90C12N2501/603C12N2501/602C12N2501/604C12N2501/727C12N2501/065C12N2501/01C12N2501/235C12N2501/15C12N2500/02C12N2500/38C12N2501/41C12N2501/70C12N2501/73C12N2510/00C12N15/87C12N2501/999
Inventor 刘承权姜泌俊孙多练洪源晙尹愿真李章辅朴圭万金仁勇朴正贤郑杰高威威张志勳全恩暻尹炳善
Owner STEMLAB
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