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Method for directly transdifferentiating mesenchymal stem cells into sperms by using transcription factor FOXO1

A technology of mesenchymal stem cells and transcription factors, applied in the field of using transcription factor FOXO1 to directly transdifferentiate mesenchymal stem cells into sperm, can solve the problem of failure to obtain mature male germ cells, and to detect the markers that cells enter the meiotic stage, etc. question

Inactive Publication Date: 2020-06-12
SHENZHEN CHILDRENS HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] In summary, in the various culture conditions or induction schemes that can promote the transdifferentiation of mesenchymal stem cells to SSCs, the markers that can be detected are limited to the early markers of male germ cells, and have not yet been detected. To the markers of cells entering the meiosis stage, there is also failure to obtain mature and functional male germ cells

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  • Method for directly transdifferentiating mesenchymal stem cells into sperms by using transcription factor FOXO1
  • Method for directly transdifferentiating mesenchymal stem cells into sperms by using transcription factor FOXO1
  • Method for directly transdifferentiating mesenchymal stem cells into sperms by using transcription factor FOXO1

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Embodiment 1

[0055] This embodiment provides a method for introducing the transcription factor FOXO1 into the mesenchymal stem cells through lentiviral transfection, including the following steps:

[0056] The green fluorescence control lentivirus (rLV-ZsGreen control lentivirus) was used to infect HuMSCs, the infection efficiency of the cells was observed, and the most suitable infection conditions were found.

[0057] (1) Prepare target cells

[0058] 1) Digest the HuMSCs that have proliferated to 80%-90% in the T75 flask with 0.05% trypsin. When the cells are digested and shrink to a round shape under a microscope, immediately add complete medium containing 10% FBS to stop the digestion, and collect the cells into a 15mL centrifuge tube.

[0059] 2) Centrifuge at 1000rpm for 5min, remove the supernatant, add fresh complete medium and gently blow off the cells, count on a hemocytometer, and make 3-5×10 4 cells / mL cell suspension, inoculate into the culture plate and continue to culture...

Embodiment 2

[0070] This embodiment provides a method for introducing the transcription factor FOXO1 into the mesenchymal stem cells by means of electroporation, including the following steps:

[0071] (1) Select HuMSCs of 3-5 generations to ensure that they are in the logarithmic growth phase (because the cell division in the logarithmic growth phase is vigorous, the surface structure is less compact, and after cell electrotransfection, foreign DNA is more likely to enter the mitotic phase cells), digest the cells with trypsin, collect the cells after termination of digestion; centrifuge at 1000g for 5 minutes, discard the supernatant, add EP buffer, resuspend the cells, continue to centrifuge, and wash 2-3 times with EP buffer, and finally resuspend For cells, the purpose of repeated washing is to effectively wash the serum on the cell surface and prevent the reduction of transfection efficiency, but the washing process should be gentle to avoid damaging the cells.

[0072] (2) Cell coun...

Embodiment 3

[0081] This embodiment provides a method for introducing the transcription factor FOXO1 into the mesenchymal stem cells by lipofection, comprising the following steps:

[0082] (1) Inoculate a certain number of HuMSCs cells into a 6-well plate, and perform transfection when the cell density reaches about 70%.

[0083] (2) Prepare the transfection mixture:

[0084] Mixture A: 4μl-8μl Lipofectamine3000+250μl Opti-MEM medium;

[0085] Mixture B: 3 μg plasmid carrying FOXO1 gene + 6 μl P3000TM + 250 μl Opti-MEM medium.

[0086] Mixture C: Mix Mixture A and Mixture B thoroughly, avoid repeated pipetting, because it will damage the DNA-liposome complex and reduce the transfection efficiency. Let stand at room temperature for 10-15 minutes to form a stable DNA-liposome complex.

[0087] (4) Transfection: Because starvation of cells can improve transfection efficiency, transfection mixture C was added to cells that had been starved for 1 hour, and then the cells were placed in a 37...

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Abstract

The invention provides a method for directly transdifferentiating mesenchymal stem cells into sperms by using a transcription factor FOXO1, comprising the following specific steps: introducing the transcription factor FOXO1 into the mesenchymal stem cells in a transfection mode, and carrying out induced culture on the mesenchymal stem cells to obtain the sperms. According to the method, the transcription factor FOXO1 is introduced into mesenchymal stem cells, and the mesenchymal stem cells are induced to develop towards sperms, so that not only can a regulatory mechanism of the stem cells on the vital movement of the organism be known, but also a signal path for maintaining the dynamic balance of the spermatogonial stem cells SSCs is discussed, and a new scheme for inheriting biological information of a male infertility patient, especially a male azoospermia patient, is provided.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for directly transdifferentiating mesenchymal stem cells into sperm by using transcription factor FOXO1. Background technique [0002] Infertility is a global medical and social problem. About 10%-20% of couples of childbearing age in the world suffer from infertility, of which male factors account for about 50%, mainly manifested as azoospermia and sperm Lack of vitality. [0003] For infertile patients with spermatogenesis defects, existing treatments such as drugs, surgery, and assisted reproductive technology are still unable to solve their fertility problems. Therefore, to study the regulatory mechanism of related molecules in the process of spermatogenesis and development, and to find mesenchymal stem cells derived from patients themselves to develop into functional germ cells for the precise treatment of male infertility is the current goal of male infertility treatm...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/867C12N3/00C12N1/14C12R1/885
CPCC12N1/14C12N3/00C12N5/061C12N15/86C12N2740/15043
Inventor 马廉
Owner SHENZHEN CHILDRENS HOSPITAL
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