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Multipotent stem cells carrying human adult premature senility syndrome gene mutations and preparation method thereof

A technology for human pluripotent stem cells and progeria, which is applied in the field of pluripotent stem cells carrying human adult progeria gene mutations and the field of preparation, can solve the problems of pluripotent stem cells that do not produce WS, and the failure to carry out efficient drug screening work, etc. To achieve the effect of delaying natural aging

Active Publication Date: 2015-06-24
INSITUTE OF BIOPHYSICS CHINESE ACADEMY OF SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] It can be seen that none of the existing reports have produced WS-PSCs and used the generated WS-PSCs to effectively simulate the disease phenotype and establish a drug screening platform, thus failing to carry out efficient drug screening work

Method used

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  • Multipotent stem cells carrying human adult premature senility syndrome gene mutations and preparation method thereof
  • Multipotent stem cells carrying human adult premature senility syndrome gene mutations and preparation method thereof
  • Multipotent stem cells carrying human adult premature senility syndrome gene mutations and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0083] Example 1, establishment and identification of WS pluripotent stem cell line

[0084] The present invention relates to targeted inactivation of human progeria gene—WRN gene in human embryonic stem cells (hESCs) (genome sequence is GenBank: 31033262-31173761 of NC_000008.11, updated on21-Mar-2015; cDNA sequence is GenBank: NM_000553.4, updated on PRI 15-MAR-2015). The present invention first designs and obtains the gene fragment spanning No. 15 and No. 16 exons of the WRN gene in the human genome through molecular cloning methods, which contains the designed mutation site and the Neo resistance gene ( figure 1 A and B in A), and then construct it into a non-integrating adenovirus vector (HDAdV vector) and infect hESCs, use the principle of homologous recombination to introduce the mutated WRN gene fragment into hESCs and replace the wild-type gene fragment, so as to realize WRN In situ inactivation of genes. Subsequent screening for Neo resistance and confirmation by g...

Embodiment 2

[0118] Example 2. WRN-deleted (WRN- / -) ESCs were differentiated in vitro to generate WRN- / - MSCs

[0119] In the present invention, the WRN- / -ESCs obtained in Example 1 were further differentiated into mesenchymal stem cells (WRN- / -MSCs) in vitro, and it was found that WRN- / -MSCs can exhibit typical symptoms of progeria WS. The specific method is as follows:

[0120] 1. Directed differentiation of mesenchymal stem cells

[0121] The WRN- / -ESCs were differentiated into embryoid bodies (EBs) for 14 days, and the EBs were seeded in matrigel-coated 6-well plates for culture, and cultured for 2 weeks until fibrous cells appeared. After another passaging, the cell populations positive for CD73, CD90, and CD105 were sorted by flow cytometry, which were WRN-deleted (WRN- / -) mesenchymal stem cells (denoted as WRN- / - MSCs). ). At the same time, a control was set in which wild-type hESCs (H9) were used instead of WRN- / -ESCs, and the resulting mesenchymal stem cells were recorded as WR...

Embodiment 3

[0137] Example 3. Using WRN- / -MSCs to screen small molecular compounds for anti-aging phenotypes

[0138] In the process of cultivating the aforementioned WRN- / -MSCs derived from WRN- / -ESCs, the present invention establishes a screening method for small molecular compounds targeting anti-aging phenotypes, and finds that vitamin C (Vitamin C, Vc) can reverse WRN- / -MSCs. - Senescent phenotype of MSCs. details as follows:

[0139] WRN-deleted MSC cells (WRN- / -MSC) were divided into 5×10 4 Spread each well in a six-well plate. From the second day after the cells adhere to the wall, add a medium containing 50M vitamin C to the experimental group to continue culturing. After culturing in a carbon dioxide culture at 37°C for about a week, the cells can grow to full size for analysis. or passage.

[0140] (1) SA-beta-Gal staining

[0141] For the specific method, refer to the above.

[0142] (2) Immunofluorescence detection

[0143] The specific method is carried out as describe...

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Abstract

The invention discloses multipotent stem cells carrying human adult premature senility syndrome gene mutations, mesenchymal stem cells and a preparation method thereof. The method comprises the following steps: (1) taking human multipotent stem cells cultured in vitro, and mutating human adult premature senility syndrome genes WRN in the human multipotent stem cells to enable the WRN to lose functions, so as to obtain the human multipotent stem cells with the function-lost WRN; and (2) carrying out induced directional differentiation on the human multipotent stem cells with the function-lost WRN to obtain the mesenchymal stem cells with the function-lost WRN. The mesenchymal stem cells derived from the multipotent stem cells provided by the invention can be used for establishing a medicine screening platform for screening a WS treatment medicine, and providing more clues for delaying a natural senility process.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a pluripotent stem cell carrying human progeria gene mutation and a preparation method. Background technique [0002] Population aging is an increasingly severe social problem facing the world, especially China. Undoubtedly, aging and its related diseases have become hot issues that urgently need breakthroughs in scientific research at home and abroad. The limitations of model organisms make it impossible to apply to the basic research and clinical transformation of human aging at the theoretical and practical levels, so new research models of human aging are urgently needed. The discovery of human "progeria" has made it possible to study human aging in the laboratory. As an extremely rare congenital genetic disease, progeria is mainly manifested as premature aging of the body, accompanied by a variety of diseases in the elderly population. It is a natural "mutation" with accelerated...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/861C12N15/55C12N5/10C12Q1/02
Inventor 刘光慧张维琦曲静任若通汤富酬铃木敬一郎胡安·卡洛斯·伊斯毕华·贝尔蒙特
Owner INSITUTE OF BIOPHYSICS CHINESE ACADEMY OF SCIENCES
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