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Directionally-inducible and anti-apoptosis pluripotent stem cells and preparation method and application thereof

A pluripotent stem cell, directional induction technology, applied in the direction of artificially induced pluripotent cells, genetically modified cells, biochemical equipment and methods, etc., can solve the problem that transplanted cells are not easy to survive, the direction of differentiation is uncontrollable, and the effectiveness cannot be obtained. Guarantee and other issues, to achieve the effect of high induction efficiency, solving safety and effectiveness issues, and wide sources

Pending Publication Date: 2020-02-21
WUYI UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] iPSCs proliferate rapidly, come from a wide range of sources, have high differentiation potential, and have no ethical controversy compared with ES. However, iPSC-based therapy still faces two major problems: on the one hand, iPSCs have a tendency to form tumors, and their safety is greatly challenged; On the one hand, the transplanted cells are not easy to survive in the body, and the direction of differentiation is uncontrollable, so its effectiveness cannot be guaranteed
Moreover, most of the directed differentiation of motor neurons is currently limited to in vitro studies, but it is not yet known whether gene-modified iPSCs can be directed and efficiently differentiated into motor neurons in vivo and have a therapeutic effect on ALS

Method used

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  • Directionally-inducible and anti-apoptosis pluripotent stem cells and preparation method and application thereof
  • Directionally-inducible and anti-apoptosis pluripotent stem cells and preparation method and application thereof
  • Directionally-inducible and anti-apoptosis pluripotent stem cells and preparation method and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Embodiment 1 of the present invention is to obtain a directional-inducible, anti-apoptotic pluripotent stem cell, the specific operation of which is as follows:

[0041] 1. Construction of Piggy-Bac (PB) vector:

[0042] (1) PB vector for inducing directed differentiation of motor neurons ①PB-Ngn2-Isl1-Lhx3(NIL)-BSD, the PB vector used in this example was donated by Italian professor Alessandro Rosa for his contribution, and the plasmid was introduced into tetracycline response factor (tetracycline response element, TRE) regulates the gene expression of Ngn2, Isl1 and Lhx3, and contains blasticidin (BSD) resistance, which can be used for cell selection.

[0043] TRE consists of seven repeated Tet operator (Tet operator, TetO) sequences.

[0044] (2) In order to improve the retention rate of hiPSCs in vivo, we constructed a PB vector ② PB-Bcl-luciferase-GFP, cloned the anti-apoptotic gene Bcl into the vector ②, and added the marker gene luciferase (luciferase) and green...

Embodiment 2

[0064]Embodiment 2 of the present invention is: an in vitro experiment of directional-induced, anti-apoptotic pluripotent stem cells, the specific operation of which is as follows:

[0065] 1. In vitro directed differentiation and identification of hiPSC-NIL-BSD+Bcl-luciferase-GFP:

[0066] (1) Induction of motor neurons: doxycycline (DOX, 1 μg / mL) was added to induce the directed differentiation of hiPSC-NIL-BSD+Bcl-luciferase+GFP.

[0067] Doxycycline DOX is a derivative of tetracycline. The inducing drug more used in the Tet-induced regulation system is DOX. Compared with tetracycline Tet, the amount of DOX required to fully activate or inhibit the expression of the target gene is less and the half-life of DOX is longer. Tetracycline drugs in this regimen can be tetracycline or its derivatives, and the DOX induction effect is better. The Tet-On system will activate transcription in the presence of tetracycline, and the transcribed Ngn2, Isl1, and Lhx3 transcription factor...

Embodiment 3

[0076] Embodiment 3 of the present invention is: an in vivo experiment of directional-induced, anti-apoptotic pluripotent stem cells, and its specific operation is as follows:

[0077] 1. Directed differentiation of hiPSC-NIL-BSD+Bcl-luciferase-GFP in vivo:

[0078] (1) HiPSC-NIL-BSD+Bcl-luciferase-GFP cell transplantation and in vivo induction: hiPSC-NIL-BSD+Bcl-luciferase-GFP (1×10 6 The cells were resuspended in 200 μL of DMEM / F12: Matrigle=1:1) and injected subcutaneously into immunodeficient mice (Severe Combined Immunodeficiency, SCID) (5-7 weeks old). Three mice in the same batch were intraperitoneally injected with DOX ( 50mg / kg), continuous injection for 7 days for directional induction.

[0079] 2. Identification of directed differentiation in vivo:

[0080] (1) Survival of hiPSCs in vivo: The luciferase signal was detected by in vivo imaging experiments, and then the survival of hiPSC-NIL-BSD+Bcl-luciferase-GFP in vivo was judged.

[0081] The obtained in vivo su...

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Abstract

The invention discloses directionally-inducible and anti-apoptosis pluripotent stem cells and a preparation method and application of the directionally-inducible and anti-apoptosis pluripotent stem cells. By utilizing a gene modification technology, a Tet-On system and an anti-apoptosis gene Bcl are transferred into hiPSC, and the hiPSC capable of being directionally induced into motor neurons isestablished. The obtained pluripotent stem cells not only retain the advantages of wide source and fast proliferation of hiPSC, but also ensure the controllability of the stem cell differentiation direction and the survival number of transplanted cells in vivo, and solve the problems of stem cell transplantation safety and effectiveness. Meanwhile, the scheme provided by the invention can lay a foundation for the treatment of ALS and other neurodegenerative diseases.

Description

technical field [0001] The invention relates to the technical field of stem cells, in particular to a directionally inducible, anti-apoptosis pluripotent stem cell and its preparation method and application. Background technique [0002] Degenerative diseases are usually accompanied by lesions or injuries of specific neurons, among which patients with amyotrophic lateral sclerosis (ALS) are mainly due to the extensive death of motor neurons. Due to the non-renewable nature of neurons, none of the current treatments can reverse nerve damage. Stem cells can be differentiated into corresponding nerve cells to replace damaged neurons and repair neural circuits, so they can be applied to the repair treatment of neurodegenerative diseases. As early as 2001, Mazzini et al. first confirmed the high safety and tolerance of mesenchymal stem cells in ALS patients in clinical trials [1] . Subsequently, more and more clinical studies pointed out that the transplantation of mesenchymal...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/85A61K35/545A61P25/02A61P21/00
CPCC12N5/0696C12N15/85C12N5/0619A61K35/545A61P25/02A61P21/00C12N2510/00C12N2800/107C12N2506/45
Inventor 陈敏王霞周继曾陈晃耀魏愈慧蓝婷邹庆剑周小青唐成程张焜周仁平赖良学
Owner WUYI UNIV
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