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Method for efficiently inducing reprogramming of human body cells into pluripotent stem cells

A technology for pluripotent stem cells and somatic cells, which is applied in the field of efficiently inducing reprogramming of somatic cells into pluripotent stem cells, which can solve the problems of long induction time, low efficiency, and restrictions on the application of iPSC research, and achieve shortened induction cycle and pluripotent gene expression uniform effect

Active Publication Date: 2011-06-15
SHENZHEN BEIKE BIOTECH
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  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Although iPSC attracts researchers from all over the world to carry out a lot of research, the existing technical system for inducing human or primate somatic cells into iPSC faces the following problems: ① its efficiency is extremely low, about 1 / 10000; ② the induction time is long, about 1 / 10000; about 24 days
The above-mentioned technical problems largely limit the research application of iPSC

Method used

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  • Method for efficiently inducing reprogramming of human body cells into pluripotent stem cells
  • Method for efficiently inducing reprogramming of human body cells into pluripotent stem cells
  • Method for efficiently inducing reprogramming of human body cells into pluripotent stem cells

Examples

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Effect test

Embodiment 1

[0045] Example 1. Amplification of retroviral vectors containing coding sequences for nuclear reprogramming factors.

[0046] According to the prior art, retroviral vectors containing human Oct3 / 4, Sox2, Klf4, and c-Myc were purchased from Addgene Company. After amplification in E. coli, retroviral vectors containing the corresponding genes were purified.

Embodiment 2

[0047] Example 2. Packaging retroviruses.

[0048] According to the prior art, retroviral vectors containing Oct3 / 4, Sox2, Klf4, and c-Myc genes are transfected into 293T cells, and the retroviruses are packaged. And pass through a 30-100KD ultrafiltration tube, 5000×g, 4°C, centrifuge for 30 minutes, collect the virus liquid, make it concentrated about 10 times, and the final concentration is about 5×10 6 -5×10 7 transducing units / mL / single nuclear reprogramming factor. Mix equal volumes of concentrated retroviruses containing Oct3 / 4, Sox2, Klf4, and c-Myc genes respectively, and take 0.6-1.0 ml of virus solution to infect somatic cells. At this time, the virus titer MOI (multiplicity of infection) is about 5-50 / single nuclear reprogramming factor.

Embodiment 3

[0049] Example 3. Amplification of lentiviral vectors containing nuclear reprogramming factor coding sequences.

[0050] According to the prior art, human Oct3 / 4, Sox2, Nanog and Lin28 lentiviral plasmids were purchased from Addgene. After amplification in E. coli, lentiviral plasmids containing the corresponding genes were purified.

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Abstract

The invention provides a method for efficiently inducing reprogramming of human body cells into pluripotent stem cells, belonging to the field of biomedicine. The method provided by the invention comprises the following step: before carrying out nuclear reprogramming on body cells, culturing the body cells for 2-4 days in a culture medium containing thyroid hormone T3. The method provided by the invention can increase the iPSC (induced pluripotent stem cell) induction efficiency by 20-100 times to about 1.2%, and shorten the induction cycle to 12-16 days; and meanwhile, different clones of the iPSC obtained by induction have uniform cellular morphology, multiplication capacity and pluripotent gene expression, and can be used for researching human diseases.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to a method for efficiently inducing reprogramming of somatic cells into pluripotent stem cells. Background technique [0002] The development and differentiation of stem cells to adult cells are precisely regulated at the gene level, and reprogramming is the reversal of the differentiation state of adult cells to the state of pluripotent stem cells. In 2007, scientists in Japan and the United States (Takahashi et al. 2007; Yu et al. 2007; Park et al. 2008) successively reported that four transcription factor genes were overexpressed by retrovirus (or lentivirus) in human somatic cells (Oct4, Sox2, Klf4 and c-Myc, or Oct4, Sox2, Nanog and Lin28), reverse the differentiation state of human adult cells, and obtain pluripotent potential stem cells (iPSC, induced pluripotent stem cell). It has been reported that somatic cells can be reprogrammed into pluripotent stem cells by introducing two...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/85
Inventor 李陶陈梦飞许笛吴捷何毅欣徐亮周欣安钟姚周向军姜舒胡祥
Owner SHENZHEN BEIKE BIOTECH
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