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Three-dimensional inducing method for inducing spermatogonia stem cell to differentiate into functional sperm cell in vitro

A technology of spermatogonial stem cells and sperm cells, which is applied in the field of three-dimensional induction of human spermatogonial stem cells differentiated into functional sperm cells in vitro, can solve the problems of inability to determine whether sperm cells have functions, no functional detection, and no clear induction efficiency.

Active Publication Date: 2017-02-22
何祖平
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Moreover, it has the following deficiencies: first, since CD49F is not a specific surface marker of human spermatogonial stem cells, the purity of the starting cells is not enough; second, only spermatozoa are detected, and there is no clear induction efficiency; third, no functional testing is performed, Unsure whether induced sperm cells are functional

Method used

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  • Three-dimensional inducing method for inducing spermatogonia stem cell to differentiate into functional sperm cell in vitro
  • Three-dimensional inducing method for inducing spermatogonia stem cell to differentiate into functional sperm cell in vitro
  • Three-dimensional inducing method for inducing spermatogonia stem cell to differentiate into functional sperm cell in vitro

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0090] A three-dimensional induction method for human spermatogonial stem cells differentiated into functional sperm cells in vitro, comprising the following steps:

[0091] 1) Weigh 1.0g of testicular tissue and put it in 10mL DMEM / F12, cut it into a volume of about 3 × 3 × 3 mm 3 Small tissue pieces, washed 3 times with DMEM / F12 to remove residual blood cells;

[0092] 2) Use scissors to cut the testicular tissue to a semi-liquid state, add 20mL DMEM / F12, and then transfer the tissue to a 120mL container;

[0093] 3) Add enzyme digestion solution Ⅰ to the testicular tissue in step 2), place in a water bath shaker at 34°C, and incubate for 10-15 minutes. The enzyme solution Ⅰ has a final concentration of 2 mg / mL collagenase solution and 1 µg / µL Composed of DNase I, which is filtered and sterilized after preparation, wherein the collagenase is type IV collagenase;

[0094] 4) Microscopically check to ensure that all tissue pieces have been digested into single seminiferous t...

Embodiment 2

[0121] Identify the sperm cells induced by Example 1 by immunocytochemical method, the steps are as follows:

[0122] a) Smear the collected haploid cells by centrifugation at 1000rpm for 5min;

[0123] b) At room temperature, wash the cell smear twice with PBS, 3min each time, and block with 200µl 10% normal goat serum for 30min;

[0124] c) Dilute the primary antibody with 100 µL PBS at 1:200 and incubate at 34°C for 1 hour or overnight at 4°C. The primary antibody is ACROSIN, PRM2, normal rabbit IgG or mouse IgG, and the negative control is replaced by PBS. ;

[0125] d) After incubation, the cells were washed 3 times with PBS, 200 µL each time;

[0126] e) Cells were incubated with fluorescein (FITC)-labeled goat anti-rabbit IgG or rhodamine-labeled goat anti-rabbit or rhodamine-labeled goat anti-mouse IgG secondary antibody for 1 hour, and stained with DAPI for nuclei, and observed under a fluorescent microscope cell.

[0127] Such as figure 2 Shown: This figure sho...

Embodiment 3

[0129] The number of chromosomes in sperm cells induced by Example 1 was detected by fluorescence in situ hybridization (FISH), and the steps were as follows:

[0130] A) Add the collected haploid cells into the fixative solution, blow evenly and let it stand for 10 minutes. The fixative solution is a mixture of methanol and acetic acid at a volume ratio of 3:1;

[0131] B) Centrifuge at 1500rpm for 10min, remove the supernatant, add fixative again, blow evenly and let stand for 10min;

[0132] C) Centrifuge at 1500rpm for 10min, remove the supernatant, and leave 20 µL of liquid;

[0133] D) Blow the cells in step C) evenly, drop onto the glass slide, and bake the slice at 56°C for 25 minutes;

[0134] E) Put the fragments of step D) into the following liquids and soak in sequence: 1mol / L sodium hydroxide for 2 minutes, 2×SSC for 10 minutes, 75% ethanol for 2 minutes, 85% ethanol for 2 minutes, and 100% ethanol for 2 minutes;

[0135] F) Dry the slide in step E), add 10 µL o...

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Abstract

The invention relates to a three-dimensional inducing method for inducing a spermatogonia stem cell to differentiate into a functional sperm cell in vitro, and belongs to the field of bioengineering. On the basis of acquiring the spermatogonia stem cell, a three-dimensional culture system is established through Matrigel, a key factor for inducing differentiation is added, the spermatogonia stem cell is induced to differentiate in vitro to obtain the sperm cell having a fertilization ability, the sperm cell obtained through induction is identified through an immunocytochemical method, the chromosome number of the sperm cell obtained through induction is detected through fluorescence in situ hybridization (FISH), and the fertilization ability and the viability of the sperm cell obtained through induction are detected through round spermatid injection (ROSI). The application provides an excellent model for the molecular mechanism of the human sperm, and in addition, a functional gamete is provided for an azoospermia patient, so that a male infertility patient can have a child of his own.

Description

technical field [0001] A three-dimensional induction method for in vitro differentiation of human spermatogonial stem cells into functional spermatids. Background technique [0002] At present, about 15% of couples of childbearing age in the world are infertile, of which male factors account for 50%; research shows that 20% of male infertility patients in Europe are azoospermia. Azoospermia is as high as 25% in Chinese male infertility patients. Moreover, due to social problems such as "late marriage and late childbearing", this disease is getting worse year by year. Therefore, obtaining germ cells with fertilization ability in vitro has always been a difficult problem and hot spot in the field of reproductive biology and reproductive medicine. Spermatogenesis refers to the process in which spermatogonial stem cells (Spermatogonial stem cells, SSCs) self-renew and differentiate into mature sperm cells. Many azoospermic patients have spermatogonial stem cells, but due to t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/076
CPCC12N5/061C12N2500/38C12N2501/125C12N2501/392C12N2513/00
Inventor 何祖平孙敏袁青青牛明辉王洪文丽平付红勇周帆李铮
Owner 何祖平
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