Transdifferentiation method of mesenchymal stem cells into sperms

A mesenchymal stem cell and transdifferentiation technology, applied in the biological field, can solve the problems of undetected markers of cells entering the meiosis stage, failure to obtain mature male germ cells, etc.

Inactive Publication Date: 2020-06-09
SHENZHEN CHILDRENS HOSPITAL
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0007] 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 marker of cells entering the meiotic stage, also failed to obtain mature and functional male germ cells

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  • Transdifferentiation method of mesenchymal stem cells into sperms
  • Transdifferentiation method of mesenchymal stem cells into sperms
  • Transdifferentiation method of mesenchymal stem cells into sperms

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Embodiment 1

[0054] This embodiment provides a method for introducing the transcription factor FOXO1 into the mesenchymal stem cells by means of lentiviral transfection, comprising the following steps:

[0055] 1. Lentiviral transfection pre-experiment:

[0056] Choose the green fluorescence control lentivirus (rLV-ZsGreen control lentivirus) to infect HuMSCs, observe the infection efficiency of the cells, and find the most suitable infection conditions.

[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 digest 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 to make 3-5×10 4 cells / mL cell suspension, inoculated into the...

Embodiment 2

[0070] This embodiment provides a method for introducing the transcription factor FOXO1 into the mesenchymal stem cells by means of electroporation, comprising 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 cou...

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 Lipofectamine 3000+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. Allow to 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, add transfection mixture C to the cells that have been starved for 1 hour, and then place the cells in a 37°...

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Abstract

The invention provides a transdifferentiation method of mesenchymal stem cells into sperms. The transdifferentiation method of mesenchymal stem cells into sperms comprises specific steps of guiding transcription factors FOXO1 into mesenchymal stem cells in a transfection manner, performing culturing to obtain stably-transfected strain, transforming culture mediums into inducing culture mediums, and continuing performing induced culture so as to obtain the sperms. According to the method disclosed by the invention, the transcription factors FOXO1 are guided into the mesenchymal stem cells, andthe inducing culture medium are used for inducing the development of the mesenchymal stem cells towards the direction of the sperms, so that the gene expression quantity of male germ cells at different stages can be increased. Therefore, the method not only contributes to understanding the adjusting and controlling mechanism of stem cells on vital movement of organisms and discussing a signal channel for maintaining dynamic equilibrium of stem spermatogonium SSCs, and a new scheme inheriting own biology information is brought for treatment of patients suffering from male infertility particularly male azoospermia.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for transdifferentiating mesenchymal stem cells into sperm. Background technique [0002] Infertility is a global medical and social problem. About 10%-15% of couples of childbearing age in the world suffer from infertility, of which male factors account for about 50%. The World Health Organization predicts that infertility, cancer, and cardiovascular disease will become the three most serious diseases that endanger human health in the 21st century. [0003] In recent years, with the progress of drug treatment and the promotion and maturity of assisted reproductive technology (Assisted reproductive technology, ART), many infertile couples have realized their dream of becoming parents. However, many infertile male patients have not been fundamentally treated, and a considerable number of infertile patients cannot solve their fertility problems through traditional drug treatm...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/076C12N5/10
CPCC12N5/061C12N2500/32C12N2500/38C12N2500/44C12N2501/115C12N2501/125C12N2501/13C12N2501/235C12N2501/60
Inventor 马廉
Owner SHENZHEN CHILDRENS HOSPITAL
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