Method for directly transdifferentiating prepuce-derived mesenchymal stem cells into sperms by using transcription factor ID4

A technology of stem cells and transcription factors, applied in the field of biomedicine, can solve the problem of not using male autologous adult stem cells

Pending Publication Date: 2022-02-08
SHENZHEN CHILDRENS HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

There have been previous studies on the differentiation of adult stem cells into male germ cells, but no studies using male autologous adult stem cells

Method used

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  • Method for directly transdifferentiating prepuce-derived mesenchymal stem cells into sperms by using transcription factor ID4
  • Method for directly transdifferentiating prepuce-derived mesenchymal stem cells into sperms by using transcription factor ID4
  • Method for directly transdifferentiating prepuce-derived mesenchymal stem cells into sperms by using transcription factor ID4

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Experimental program
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Effect test

Embodiment 1

[0055] This embodiment provides a method of using transcription factor ID4 to directly transdifferentiate foreskin-derived mesenchymal stem cells into sperm, which includes the following steps:

[0056] Step 1, packaging and purification of LV-hID4-Hygro-puro lentivirus: a three-plasmid lentivirus system is used for the virus packaging process. The three-plasmid lentiviral system includes the following plasmids: a vector plasmid carrying a gene of interest or shRNA, a viral packaging helper plasmid (psPAX2 vector), and a viral packaging helper plasmid (pMD2G vector).

[0057] 1.1 When the confluence of 293T cells reaches about 70%, passage 293T cells for transfection. The cells to be transfected should be prepared the day before: add 1ml 0.25% EDTA trypsin to each 10cm culture dish, shake and mix gently, so that all cells are exposed to trypsin, and then place it at 37°C with carbon dioxide volume Digest in a constant temperature and humidity incubator with a fraction of 5% f...

Embodiment 2

[0131] In this embodiment, flow cytometry is used to detect the haploid yield of ID4-FSMSCs-30, and the specific experimental steps are as follows:

[0132] 1. ID4-FSMSCs were induced and cultured for 30 days, the original medium was discarded, fresh medium was added, and live cell DNA dye (Hoechst) was added to make the final concentration 1 μg / ml, placed at 37°C, 5% CO 2 Cell incubator for 1 hour.

[0133] 2. After 1 hour of staining, the cells were taken out and washed twice with PBS for 5 minutes each time.

[0134] 3. Digest the cells with 0.25% trypsin, centrifuge at 800rcf for 5min, resuspend the cells with flow cytometry working solution containing 2% FBS+DPBS and count them, divide the cells into 5ml flow cytometry tubes, 5×10 per tube 5 cells.

[0135] 4. Centrifuge at 800rcf for 5min, and suck off the supernatant. Use 200 μL of flow cytometry working solution to resuspend cells, and use BDAriaIII flow cytometer for cell flow cytometry detection.

[0136] result ...

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Abstract

The invention provides a method for directly transdifferentiating prepuce-derived mesenchymal stem cells into sperms by using a transcription factor ID4. The method comprises the following steps: introducing the transcription factor ID4 into the mesenchymal stem cells in a transfection manner, and then performing induced culture on the mesenchymal stem cells to obtain male autologous haploid germ cells (sperms). According to the method provided by the invention, the mesenchymal stem cells cultured from the male autologous prepuce are directly transdifferentiated into the male autologous haploid germ cells by using the transcription factor ID4, germ cells with male genetic codes and fertilization ability are obtained, and an effective scheme is provided for treating male infertility with spermatogenesis disorder.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to a method for directly transdifferentiating foreskin-derived mesenchymal stem cells into sperm by using transcription factor ID4. Background technique [0002] Infertility is a global medical and social problem. About 10%-20% of couples of childbearing age worldwide 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] The current method of treating human infertility is the combination of assisted reproductive technology (Assisted reproductive technology, ART) with drug therapy and surgical treatment, which has fulfilled the dream of many infertile couples to become parents. However, many infertile male patients have not been treated fundamentally, and a considerable num...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/12C12N15/867C12N5/10C12N5/076
CPCC07K14/47C12N15/86C12N5/0668C12N5/061C12N2510/00C12N2500/32C12N2500/44C12N2501/115C12N2501/13C12N2501/125C12N2501/235C12N2740/15043
Inventor 马廉
Owner SHENZHEN CHILDRENS HOSPITAL
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