A method for efficiently obtaining porcine induced pluripotent stem cells

A pluripotent stem cell and inducible technology, applied in the field of cells, can solve the problems of low efficiency and long cycle of pig iPSCs, and achieve the effect of improving efficiency, shortening cycle and improving quality

Active Publication Date: 2017-02-15
ANHUI AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0011] In order to overcome the above-mentioned problems of low efficiency and long cycle of inducing porcine iPSCs, the object of the present invention is to provide a technical method for efficiently obtaining porcine iPSCs. The method is to induce reprogramming of ADSCs under feeder-free and serum-free culture conditions Pluripotent stem cells, and cells were treated with MEK signaling pathway inhibitor PD0325901 and GSK3 signaling pathway inhibitor CHIR99021 at specific stages in the reprogramming process

Method used

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  • A method for efficiently obtaining porcine induced pluripotent stem cells
  • A method for efficiently obtaining porcine induced pluripotent stem cells
  • A method for efficiently obtaining porcine induced pluripotent stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1. Preparation and cultivation of cells

[0047] 1.1 Culture of porcine adipose stem cells (ADSCs)

[0048]Separate the adipose tissue from the pig's back and abdomen subcutaneously, remove blood vessels and muscle residues, wash thoroughly with DPBS buffer (Gibco), then use surgical scissors to fully cut the adipose tissue until there is almost no shear resistance, and transfer to the centrifuge. In the tube, add 0.09% type I collagenase digestion solution (Sigma company) equivalent to 2 to 3 times the total volume of adipose tissue, and place it in a water bath at 37°C for shaking and digestion; after the tissue suspension is gelatinized, use Pasteur The pipette was blown repeatedly to disperse tissue debris, centrifuged at room temperature at 1,200rpm for 5min, discarded the mature adipose tissue in the upper layer of the centrifuge tube and the digestive juice in the middle layer, and then used DMEM / F-12 basal medium containing 10% fetal bovine serum (HyClon...

Embodiment 2

[0053] Example 2. Infection of porcine ADSCs with viral vectors

[0054] According to the method described in Example 1, low-passage porcine ADSCs were inoculated in cell culture flasks at 37°C, 5% CO 2 When the confluence reached 80-90% under conventional culture conditions, digested with 0.25% trypsin-EDTA (Gibco) at 37°C to form a single-cell suspension, and infected 1×10 cells with the collected virus supernatant. 5 ADSCs, the multiplicity of infection (MOI) was 3 (the titer of the virus used was 5-10×10 6 IU / mL, the four viruses carrying the pluripotency factor cDNA were mixed according to 1:1:1:1), and then inoculated into 6-well culture plates, and continued to store at 37°C, 5% CO 2 cultured under conventional culture conditions. The viral supernatant was transfected into 293T cells (Fugene HD by a conventional method) with a drug-inducible (RevTet-On) lentiviral expression vector (SiDanSai Company) containing cDNA of human Oct4, Sox2, Klf4 and c-Myc. , Roche Compan...

Embodiment 3

[0055] Example 3. Continued culture and clone screening of infected cells

[0056] On the second day after infection, the culture wells were washed twice with DPBS buffer, and then the infected ADSCs were digested into a single-cell suspension with 0.25% trypsin-EDTA (Gibco Company) at 37°C, and the cells were divided into 2,500 cells. / cm 2 The density was inoculated into a new 6-well culture plate, and a total of 4 culture wells were inoculated. The surface of the culture plate was pre-coated with Matrigel (BDPharmingen Company), and ADSCs complete medium was continued to be used. Among the parallel samples of 4 wells here, 1 well was used for alkaline phosphatase (AP) staining (SiDanSai Company), and the number of positive clones was counted, and the other 3 wells were used for clone selection. The experiment was repeated three times. On the third day after infection, the ADSCs complete medium was replaced with the porcine iPSCs serum-free complete medium described in Exam...

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Abstract

The invention discloses a method for efficiently acquiring inductive pluripotent stem cells (iPSCs). Adipose-derived stem cells (ADSCs) are induced and reprogrammed to form the iPSCs by using the method. The method concretely comprises the following steps: (1) inducing cDNA (Complementary Desoxvribose Nucleic Acid) of a multifunctional factor to the ADSCs; (2) culturing the ADSCs by using a serum-free culture medium in a feed-layer-free system, wherein the ADSCs is obtained in the step (1); (3) after cloning sample cells of embryonic stem cells (ESCs), further culturing by using a culture medium with an MEK (Methyl Ethyl Ketone) and GSK3 (Glaxo Smith Klein) signal channel inhibitor, selecting cells to clone, and enlarging culture; and (4) authenticating the cell cloning pluripotency. In the method, the ADSCs are low in cost and easily available on a large scale, and the reprogramming speed and efficiency are very high; the feed-layer-free system can be used for increasing the cloning purity of positive iPSCs; the serum-free culture medium can be used for avoiding unstable factors brought by undefined components in serum and batch difference; the signal channel inhibitor can be used for promoting cells to be up to the sufficient reprogramming state and improving the cloning quality of the positive iPSCs.

Description

technical field [0001] The invention relates to the field of cells, in particular to a method for efficiently obtaining porcine induced pluripotent stem cells. Background technique [0002] Stem cells are the initial source of the human body and its various tissue cells. Their most notable biological characteristics are the ability of self-renewal and continuous proliferation, as well as the potential of multidirectional differentiation. Stem cells are divided into adult stem cells (Adult stem cells) and embryonic stem cells (Embryonic stem cells, ESCs) according to different sources. Adult stem cells include bone marrow mesenchymal stem cells, pancreatic stem cells, neural stem cells, adipose stem cells, etc., which exist in adult tissues. [0003] In 1981, the isolation and culture of ESCs was first achieved in mice, and it is the most widely studied and mature stem cell system so far. The generation of human stem cells began in 1998. American scientist James A. Thomson ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/867C12N5/10
Inventor 张运海张宇魏超章孝荣
Owner ANHUI AGRICULTURAL UNIVERSITY
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