CN113481150A - Application of EGFR inhibitor in improving reprogramming efficiency of spermatogonial stem cells - Google Patents

Application of EGFR inhibitor in improving reprogramming efficiency of spermatogonial stem cells Download PDF

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CN113481150A
CN113481150A CN202110653660.XA CN202110653660A CN113481150A CN 113481150 A CN113481150 A CN 113481150A CN 202110653660 A CN202110653660 A CN 202110653660A CN 113481150 A CN113481150 A CN 113481150A
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spermatogonial stem
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daphnetin
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赵小阳
汪妹
许言文
陈雨寒
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Southern Medical University
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Abstract

The invention belongs to the field of stem cell biology and cell reprogramming, and particularly relates to an application of an EGFR inhibitor in improving the reprogramming efficiency of spermatogonial stem cells. The invention also provides a composition comprising an EGFR inhibitor, vitamin C, SGC707, tauroursodeoxycholic acid; the EGFR inhibitor is at least one of daphnetin and epigallocatechin gallate. The composition can simply, conveniently, quickly and efficiently induce spermatogonial stem cells to reprogram into pluripotent stem cells with good differentiation potential in vitro and in vivo and germ line chimeric ability under the condition of not introducing or using any exogenous gene/transcription factor/MicroRNA (miRNA) and related inducing factors such as RNA, polypeptide or protein, and the like, and each component has good drug compatibility, low cost, good stability and simple operation.

Description

Application of EGFR inhibitor in improving reprogramming efficiency of spermatogonial stem cells
Technical Field
The invention belongs to the field of stem cell biology and cell reprogramming, and particularly relates to an application of an EGFR inhibitor in improving the reprogramming efficiency of spermatogonial stem cells.
Background
Stem cells are characterized by their ability to self-renew and to differentiate divergently, i.e., stem cells can produce cells with the same characteristics as themselves to maintain self-renewal, and can also differentiate to produce functionally specialized cells. The characteristics of the stem cells enable the stem cells to have great application value in the field of regenerative medicine.
Spermatogonial Stem Cells (SSCs), a type of adult stem cell that exists in the male testis or testis, are an important guarantee for maintaining a constant spermatogenesis process throughout the life after puberty. In the in vitro long-term culture process, the mouse spermatogonial stem cells can be spontaneously reprogrammed to become pluripotent stem cells (gPSCs), the gPSCs can be amplified and cultured in an embryonic stem cell culture solution, and the gPSCs have good in vivo and in vitro differentiation potential and germline chimeric capacity, so the method has potential application prospect.
However, the prepro-spermatogonial stem cell reprogramming system established before has the defects of low efficiency (about 0.05%), long time course (about 28 days), unstable system and the like, so that the research on the spontaneous reprogramming mechanism of the spermatogonial stem cell and the application thereof face obstacles. Therefore, the development of a system for efficiently and rapidly inducing the reprogramming of the spermatogonial stem cells is very important for the development of the field of cell reprogramming.
Disclosure of Invention
The first aspect of the invention aims to provide the application of the EGFR inhibitor in improving the reprogramming efficiency of spermatogonial stem cells.
In a second aspect, the present invention is directed to a composition.
In a third aspect, the present invention provides the use of the composition of the second aspect of the present invention for improving the reprogramming efficiency of spermatogonial stem cells.
In a fourth aspect, the present invention provides the use of the composition of the second aspect of the present invention to improve the reprogramming time of spermatogonial stem cells.
In a fifth aspect, the present invention provides a method for improving the reprogramming efficiency of spermatogonial stem cells.
In a sixth aspect, the present invention provides a method for shortening the reprogramming time of spermatogonial stem cells.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
in a first aspect of the invention, there is provided the use of an EGFR inhibitor which is at least one of daphnetin and epigallocatechin gallate for increasing the efficiency of reprogramming of spermatogonial stem cells.
Preferably, the EGFR inhibitor is daphnetin.
Preferably, the concentration of the EGFR inhibitor is 2.5-10 mu M; further preferably, the concentration of the EGFR inhibitor is 7.5-10 mu M.
In a second aspect of the invention, there is provided a composition comprising an EGFR inhibitor, vitamin C, SGC707 and tauroursodeoxycholic acid; the EGFR inhibitor is at least one of daphnetin and epigallocatechin gallate.
Preferably, the EGFR inhibitor is daphnetin and epigallocatechin gallate.
Preferably, the composition comprises the following components: daphnetin, epigallocatechin gallate, vitamin C, SGC707, and tauroursodeoxycholic acid.
Further preferably, the composition comprises the following components: 2.5-10 mu M daphnetin, 5-10 mu M epigallocatechin gallate, 283.9-380.4 mu M vitamin C, 2.5-10 mu M SGC707 and 10-20 mu M tauroursodeoxycholic acid.
Even more preferably, the composition comprises the following components: 7.5-10 mu M daphnetin, 5-7.5 mu M epigallocatechin gallate, 283.9-380.4 mu M vitamin C, 5-10 mu M SGC707 and 10-20 mu M tauroursodeoxycholic acid.
In a third aspect of the invention, there is provided the use of a composition according to the second aspect of the invention for increasing the efficiency of reprogramming spermatogonial stem cells.
In a fourth aspect of the invention, there is provided the use of a composition according to the second aspect of the invention for shortening the reprogramming time course of spermatogonial stem cells.
In a fifth aspect of the present invention, there is provided a method for increasing the reprogramming efficiency of spermatogonial stem cells, comprising mixing an EGFR inhibitor or a composition according to the third aspect of the present invention with spermatogonial stem cells, and culturing; the EGFR inhibitor is at least one of daphnetin and epigallocatechin gallate.
Preferably, the EGFR inhibitor is daphnetin.
Preferably, the concentration of the EGFR inhibitor is 2.5-10 mu M; further preferably, the concentration of the EGFR inhibitor is 7.5-10 mu M.
In a sixth aspect of the present invention, there is provided a method for shortening the reprogramming time of spermatogonial stem cells, comprising mixing the composition of the third aspect of the present invention with spermatogonial stem cells, and culturing the mixture.
The invention has the beneficial effects that:
the invention discloses application of an EGFR inhibitor (daphnetin and/or epigallocatechin gallate) in improving the reprogramming efficiency of spermatogonial stem cells for the first time, and can remarkably improve the reprogramming efficiency of the spermatogonial stem cells.
The invention provides a composition, which comprises the following components: daphnetin, epigallocatechin gallate, vitamin C, SGC707 and tauroursodeoxycholic acid; the composition can remarkably improve the reprogramming efficiency of the spermatogonial stem cells (about 100 times), and the clone number of the Oct4-GFP positive cells of the composition after the spermatogonial stem cells are treated is larger than the sum of the clone numbers of the Oct4-GFP positive cells of the composition after the spermatogonial stem cells are treated by the composition, namely the reprogramming efficiency of the spermatogonial stem cells of the composition is larger than the sum of the reprogramming efficiency of the spermatogonial stem cells of the composition, which indicates that the synergistic effect can be achieved among the components of the composition; meanwhile, the composition can improve the acquisition quantity of the pluripotent stem cells (about 1000 times), shorten the reprogramming time of the spermatogonial stem cells (about 1 time and 10 days), and induce the spermatogonial stem cells to reprogram to obtain the pluripotent stem cells with differentiation and development capacity; therefore, the composition provided by the invention can simply, conveniently, quickly and efficiently induce the spermatogonial stem cells to be reprogrammed into the pluripotent stem cells with good differentiation potential in vitro and in vivo and germ line chimeric ability under the condition of not introducing or using any exogenous gene/transcription factor/MicroRNA (miRNA) and related inducing factors such as RNA, polypeptide or protein, and the like, and each component has good pharmacy, low cost, good stability and simple operation.
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FIG. 1 is a bright field diagram of spermatogonial stem cells in example 1.
FIG. 2 is a statistical chart of the number of Oct4-GFP positive cell clones obtained by reprogramming the spermatogonial stem cells in example 1 at different concentrations of daphnetin: denotes p < 0.05.
FIG. 3 is a statistical chart of the number of Oct4-GFP positive cell clones obtained by reprogramming the spermatogonial stem cells in example 1 at different concentrations of epigallocatechin gallate: denotes p < 0.05.
FIG. 4 is a statistical chart of the number of Oct4-GFP positive cell clones obtained by reprogramming the spermatogonial stem cells in example 1 at different concentrations of vitamin C: denotes p < 0.05.
FIG. 5 is a statistical chart of the number of Oct4-GFP positive cell clones obtained by reprogramming the spermatogonial stem cells in example 1 under SGC707 at various concentrations: denotes p < 0.05.
FIG. 6 is a statistical chart of the number of Oct4-GFP positive cell clones obtained by reprogramming the spermatogonial stem cells in example 1 under different concentrations of tauroursodeoxycholic acid: denotes p < 0.05.
FIG. 7 is a statistical chart of the number of Oct4-GFP positive clones obtained by reprogramming the spermatogonial stem cells in example 2 under vitamin C and tauroursodeoxycholic acid at different concentrations: denotes p < 0.05.
FIG. 8 is a statistical chart of the number of Oct4-GFP positive clones obtained by reprogramming the spermatogonial stem cells in example 2 under vitamin C, tauroursodeoxycholic acid and daphnetin at different concentrations: denotes p < 0.05.
FIG. 9 is a statistical chart of the number of Oct4-GFP positive clones obtained by reprogramming the spermatogonial stem cells in example 2 under vitamin C, tauroursodeoxycholic acid, daphnetin and SGC707 at different concentrations: denotes p < 0.01.
FIG. 10 is a statistical chart of the number of Oct4-GFP positive clones obtained by reprogramming the spermatogonial stem cells in example 2 under vitamin C, tauroursodeoxycholic acid, daphnetin, SGC707, and epigallocatechin gallate at various concentrations: denotes p < 0.05.
FIG. 11 graph of the effect of the composition of example 2 on the reprogramming efficiency of spermatogonial stem cells: wherein A is an Oct4-GFP positive cell clone obtained by reprogramming the spermatogonial stem cells after being treated by the composition and an alkaline phosphatase staining positive clone detection result chart; b is a statistical chart of the number of Oct4-GFP positive cell clones obtained by reprogramming the spermatogonial stem cells after being treated by the composition: denotes p < 0.0001.
FIG. 12 is a graph of the effect of the composition of example 2 on the number of pluripotent stem cells obtained by reprogramming spermatogonial stem cells: wherein A is a visual map of the number of pluripotent stem cells obtained by reprogramming spermatogonial stem cells treated with the composition; b is a statistical chart of the number of pluripotent stem cells obtained by reprogramming the spermatogonial stem cells after being treated by the composition: denotes p < 0.0001.
FIG. 13 is a graph of the effect of the composition of example 2 on the time course of reprogramming of spermatogonial stem cells.
FIG. 14 is a three-germ layer differentiation graph of pluripotent stem cells obtained by inducing reprogramming of spermatogonial stem cells with the composition of example 2.
FIG. 15 is a graph showing the results of functional verification of pluripotent stem cells obtained by reprogramming spermatogonial stem cells induced by the composition of example 2: wherein A is the bright field and fluorescence map of the genital ridges of embryos 12 days after the blastocyst injection of 5 SM-gPSC; b is a picture of the F0 generation chimeric mice born after blastocyst injection of 5 SM-gPSC.
Detailed Description
The present invention will be described in further detail with reference to specific examples.
It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.
The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers. The materials, reagents and the like used in the present examples are commercially available reagents and materials unless otherwise specified.
Example 1 Effect of Daphnetin (Daphnetin), Epigallocatechin Gallate ((-) -Epigallocatechin Gallate, EGCG), vitamin C (Vc), SGC707, tauroursodeoxycholic acid (TUDCA) on reprogramming efficiency of spermatogonial stem cells
1. Establishment of spermatogonial stem cell line
1 mouse (C57BL/6x DBA) at 5.5 days after birth was removed, sacrificed by cervical dislocation, and the abdominal skin of the mouse was excised. Pulling out testis, placing in 3.5cm cell culture dish containing PBS, and removing testis white membrane in the dish; transferring the cleaned testis seminiferous tubule to a new 3.5cm cell Culture dish containing PBS, adding collagenase IV, digesting for 7 min, observing most OF the lumen under the microscope to disperse, adding pancreatin 2mL containing 0.05% EDTA, digesting for 15 min to form single cell state, stopping the digestion with DMEM containing 10% fetal calf serum, filtering with 70 μm cell sieve, resuspending with spermatogonium Stem cell Culture fluid (Kanatsu-Shinohara M. Long-Term Proliferation in Culture fluid and Germinium Transmission OF Mouse Male Germinium Stem Cells J. BIOLOGY OF RODUCTION,2003.69:5. Mero Mouse spermatogonium Stem cell Culture fluid), planting on a cell Culture plate coated with gelatin, after 24h, adhering testis body cell part to the bottom OF the Culture plate coated with gelatin due to difference, keeping most OF germ Cells in suspension state in testis, at this point, the medium was transferred to a new 12-well plate, and half of the medium was replaced every other day. After the primary culture is continued for 7 days, the protamine stem cells are observed under the mirror to be grape-shaped clones (figure 1) and grow on the upper part of somatic cells. At this time, pancreatin digestion passage can be carried out, during passage, 0.05% pancreatin is used for digestion at 37 ℃ for 2min, after digestion into single cells, the digestion is stopped by DMEM containing 10% fetal calf serum, and spermatogonial stem cells are obtained.
2. Configuration for concentrating small molecule compound
Dissolving Daphnetin (Daphnetin), Epigallocatechin Gallate ((-) -Epigallocatechin Gallate, EGCG), SGC707 and tauroursodeoxycholic acid (TUDCA) in DMSO, respectively, and dissolving vitamin C in water; under the aseptic condition, the five small molecules are prepared into a 100mM concentrated storage. For example: daphnetin dissolved 17.81mg of the powder in 1mL of DMSO to give a final concentration of 100mM in stock; ECGC dissolved 45.84mg of the powder in 1mL of DMSO to give a final concentration of 100mM in the concentrate; SGC707 dissolving 29.83mg of the powder in 1mL of DMSO to give a final concentration of 100mM in the stock; TUDCA dissolved 49.97mg of the powder in 1mL of DMSO to give a final concentration of 100mM in stock; vitamin C17.6 mg of the powder was dissolved in 1mL of water to give a final concentration of 100mM in the concentrate. After the compound is completely dissolved, subpackaging, storing in a refrigerator at-20 ℃, and adding the concentrated compound into a culture solution of spermatogonial stem cells to dilute to a specific concentration for SSC reprogramming when in use.
3. Effect of daphnetin on reprogramming efficiency of spermatogonial stem cells
The spermatogonial stem cells were inoculated into 24-well culture dishes previously prepared with Fibronectin (fibrinectin) in the amount of 4,000 cells per well, daphnetin (final concentrations of 0, 2.5, 5, 7.5, and 10 μ M, respectively, and the solvent was a culture solution of the spermatogonial stem cells) was added, each treatment was repeated 3 times (6 duplicate wells per group, 18 duplicate wells per treatment), 500 μ L per well was replaced every 4 days, and after 19 days of culture, the number of Oct4-GFP positive cell clones per well was determined (the appearance of Oct4-GFP strong positive clones was used as a sign of successful reprogramming), with the results shown in fig. 2: 2.5-10 mu M daphnetin can obviously improve the reprogramming efficiency of the primitive stem cells, and the reprogramming effect is optimal particularly when the concentration is 7.5 mu M.
4. Effect of Epigallocatechin Gallate ((-) -Epigallocatechin Gallate, EGCG) on reprogramming efficiency of spermatogonial stem cells
The spermatogonial stem cells were inoculated into 24-well culture dishes previously prepared with Fibronectin (Fibronectin) in a cell amount of 4,000 per well, EGCG (final concentrations of 0, 2.5, 5, 7.5, and 10 μ M, respectively, and the solvent was a spermatogonial stem cell culture solution) was added, each treatment was repeated 3 times (6 replicate wells per group, 18 replicate wells per treatment), 500 μ L per well was replaced every 4 days, and after 19 days of culture, the number of Oct4-GFP positive cell clones per well was determined (the occurrence of Oct4-GFP strong positive clones was used as a sign of successful reprogramming), with the results shown in fig. 3: when the concentration is 5-10 mu M, the EGCG can remarkably improve the reprogramming efficiency of the primitive stem cells.
5. Effect of vitamin C (Vc) on the reprogramming efficiency of spermatogonial stem cells
The spermatogonial stem cells were inoculated into 24-well culture dishes previously prepared with Fibronectin (Fibronectin) in a cell amount of 4,000 per well, Vc (final concentrations of 0, 113.6, 170.3, 283.9, 380.4 μ M, solvent was spermatogonial stem cell culture solution) was added to each well, each treatment was repeated 3 times (6 replicate wells per group, 18 replicate wells per treatment), 500 μ L per well was replaced every 4 days, and after 19 days of culture, the number of Oct4-GFP positive cell clones per well was determined (appearance of Oct4-GFP strong positive clones was used as a sign of successful reprogramming), with the results shown in fig. 4: when the concentration is 283.9-380.4 mu M, Vc can remarkably improve the reprogramming efficiency of the primitive stem cells.
Effect of SGC707 on efficiency of reprogramming spermatogonial stem cells
The spermatogonial stem cells were inoculated into 24-well culture dishes previously prepared with Fibronectin (Fibronectin) in the amount of 4,000 cells per well, SGC707 (final concentrations of 0, 2.5, 5, 7.5, and 10 μ M, respectively, and the solvent was a spermatogonial stem cell culture solution) was added to each well, each treatment was repeated 3 times (6 duplicate wells per group, 18 duplicate wells per treatment), 500 μ L per well was replaced every 4 days, and after 19 days of culture, the number of Oct4-GFP positive cell clones per well was determined (the appearance of Oct4-GFP strong positive clones was used as a sign of successful reprogramming), with the results shown in fig. 5: when the concentration is 2.5-10 mu M, SGC707 can remarkably improve the reprogramming efficiency of the primitive stem cells.
7. Effect of tauroursodeoxycholic acid (TUDCA) on reprogramming efficiency of spermatogonial stem cells
The spermatogonial stem cells were inoculated into 24-well culture dishes previously coated with Fibronectin (Fibronectin) in a cell amount of 4,000 per well, TUDCA (final concentrations of 0, 2.5, 5, 10, and 20 μ M, respectively, and a solvent was a culture solution of the spermatogonial stem cells) was added, each treatment was repeated 3 times (6 replicate wells per group, 18 replicate wells per treatment), 500 μ L per well was replaced every 4 days, and after 19 days of culture, the number of Oct4-GFP positive cell clones per well was determined (the occurrence of Oct4-GFP strong positive clones was used as a sign of successful reprogramming), with the results shown in fig. 6: when the concentration is 10-20 mu M, SGC707 can remarkably improve the reprogramming efficiency of the primitive stem cells.
Example 2A composition
1. Determination of the concentration of the Components (daphnetin, Epigallocatechin gallate, vitamin C, SGC707 and tauroursodeoxycholic acid) in composition (5SM)
After the optimal concentration of vitamin C (Vc) for promoting spermatogonial stem cells is determined to be 380.4 mu M, the next small molecule is added one by one on the basis, and the optimal combined concentration is tested: the spermatogonial stem cells were seeded in 24-well plates previously prepared with Fibronectin (Fibronectin) at a cell count of 4,000 spermatogonial stem cells per well, and the composition Vc (380.4 μ M final concentration) + TUDCA was added at 5.0 μ M, 10 μ M, and 20 μ M final concentrations, respectively, to the control group, in which only Vc (380.4 μ M concentration) was added. Each treatment was repeated 3 times (6 replicates per group, 18 replicates per treatment), 500 μ L per well, half fluid was changed every 4 days, and after 19 days of culture, the number of Oct4-GFP positive cell clones per well (with the appearance of Oct4-GFP strong positive clones as a sign of successful reprogramming) was examined, with the results shown in fig. 7: when the concentration of Vc is 380.4 μ M and the concentration of TUDCA is 10 μ M, the reprogramming efficiency of the primitive stem cells can be remarkably improved.
After the concentration of Vc + TUDCA was determined, the Daphnetin addition concentration was tested again on the basis of the determined concentration. The spermatogonial stem cells were seeded in 24-well plates previously prepared with Fibronectin (Fibronectin) in a cell amount of 4,000 spermatogonial stem cells per well, and the composition Vc (final concentration of 380.4 μ M) + TUDCA (final concentration of 10 μ M) + Daphnetin (final concentration of 5.0 μ M, 7.5 μ M, 10 μ M) was added to the control group, in which case Vc (final concentration of 380.4 μ M) + TUDCA (final concentration of 10 μ M) was added. Each treatment was repeated 3 times (6 replicates per group, 18 replicates per treatment), 500 μ L of liquid per well, half of the liquid was changed every 4 days, and after 19 days of culture, the number of Oct4-GFP positive cell clones per well (the appearance of Oct4-GFP strong positive clones was used as a marker for successful reprogramming) was determined, and the results are shown in fig. 8: when the concentration of Vc is 380.4 μ M and the concentration of TUDCA is 10 μ M, the concentration of Daphnetin of 7.5 μ M can significantly improve the reprogramming efficiency of the primitive stem cells.
After the concentration of Vc + TUDCA + Daphnetin was determined, the SGC707 concentration was again tested on this basis. The spermatogonial stem cells were seeded in 24-well plates previously prepared with Fibronectin (Fibronectin) in a cell amount of 4,000 spermatogonial stem cells per well, and the composition Vc (final concentration of 380.4 μ M) + TUDCA (final concentration of 10 μ M) + Daphnetin (final concentration of 10 μ M) + SGC707 was added to the plates, and the final concentration of SGC707 was 5.0 μ M, 7.5 μ M, and 10 μ M, respectively, at which time Vc (final concentration of 380.4 μ M) + TUDCA (final concentration of 10 μ M) + Daphnetin (final concentration of 10 μ M) was added to the control group. Each treatment was repeated 3 times (6 replicates per group, 18 replicates per treatment), 500 μ L of liquid per well, half of the liquid was changed every 4 days, and after 19 days of culture, the number of Oct4-GFP positive cell clones per well (the appearance of Oct4-GFP strong positive clones was used as a marker for successful reprogramming) was determined, and the results are shown in fig. 9: when the concentration of Vc is 380.4 μ M, the concentration of TUDCA is 10 μ M, and the concentration of Daphnetin is 7.5 μ M, the concentration of SGC707 is 7.5 μ M, so that the reprogramming efficiency of the primitive stem cells can be remarkably improved.
After the concentration of Vc + TUDCA + Daphnetin + SGC707 was determined, the concentration of (-) -Epigallocatachin Gallate was tested based on the determined concentration. The spermatogonial stem cells were seeded in 24-well plates previously coated with Fibronectin (Fibronectin) in an amount of 4,000 cells per well, and the compositions Vc (final concentration of 380.4 μ M) + TUDCA (final concentration of 10 μ M) + Daphnetin (final concentration of 10 μ M) + SGC707 (final concentration of 7.5 μ M) + (-) -Epigallocatechin Gallate, (-) -Epigallocatechin Gallate were added at final concentrations of 5.0 μ M, 7.5 μ M and 10 μ M, respectively, in which case Vc (final concentration of 380.4 μ M) + TUnetDCA (final concentration of 10 μ M) + Daphin (final concentration of 10 μ M) + SGC707 (final concentration of 7.5 μ M) were added to the control group. Each treatment was repeated 3 times (6 replicates per group, 18 replicates per treatment), 500 μ L of liquid per well, half of the liquid was changed every 4 days, and after 19 days of culture, the number of Oct4-GFP positive cell clones per well (the appearance of Oct4-GFP strong positive clones was used as a marker for successful reprogramming) was determined, and the results are shown in fig. 10: when the concentration of Vc is 380.4 muM, the concentration of TUDCA is 10 muM, the concentration of Daphnetin is 7.5 muM, and the concentration of SGC707 is 7.5 muM, (-) -Epigallocatechin Gallate concentration is 7.5 muM, the reprogramming efficiency of the protostem cells can be remarkably improved.
2. A composition (5SM) consisting of: daphnetin (Daphnetin) 7.5. mu.M, Epigallocatechin Gallate ((-) -Epigallocatechin Gallate, EGCG) 7.5. mu.M, vitamin C (Vc) 67. mu.g/mL, SGC 7077.5. mu.M and tauroursodeoxycholic acid (TUDCA) 10. mu.M; the preparation method comprises the following steps: the compounds of example 1 were taken out and concentrated, and added to the spermatogonial stem cell culture medium to obtain the final product.
3. Effect of composition (5SM) on efficiency of reprogramming of spermatogonial Stem cells
The spermatogonial stem cells were inoculated into 24-well plates previously coated with Fibronectin (Fibronectin) in the amount of 4,000 cells per well, the composition (5SM) in 1 was added (control group, solvent in equal amount of concentrated stock was added to the culture solution of the spermatogonial stem cells according to the above composition preparation method), 6 wells were each repeated 3 times (18 wells for each treatment), 500 μ L of the half solution was replaced every 4 days, and after culturing for 19 days, the number of Oct4-GFP positive cell clones (the appearance of Oct4-GFP strong positive clones was used as a sign of successful reprogramming) and Alkaline phosphatase (aikaline phosphate, AP) staining positive clones per well were examined, as shown in fig. 11: the composition (5SM) can significantly improve the reprogramming efficiency of the primitive stem cells, wherein the number of Oct4-GFP positive cell clones (about 90) in the composition (5SM) is about 100 times that of the control group (DMSO), and is greater than the sum of the number of Oct4-GFP positive cell clones (4+9+13+10+ 12-48) in each component, indicating that each component in the composition (5SM) produces a synergistic effect.
4. Effect of composition (5SM) on the number of pluripotent Stem cells (gPSCs) obtained
The spermatogonial stem cells were seeded in a 24-well plate previously coated with Fibronectin (Fibronectin) in a cell amount of 4,000 per well, the composition (5SM) in 1 was added (control group prepared by the above composition method, the solvent in an equal amount of concentrate was added to the spermatogonial stem cell culture solution in a concentration negligible to the toxicity of the spermatogonial stem cells), 6 wells were added for each group, each treatment was repeated 3 times (18 wells for each treatment), 500. mu.L of the half solution was replaced every 4 days, and after culturing for 19 days, all the cells in the wells were digested and passaged to an embryonic stem cell culture solution containing N2B27+ CHIR99021+ PD 5901+ mLIF2i (specific formulation reference: Ying QL, Wray J, Nichols J, et al, the group of embryonic stem cells selected in cell culture solution [ J6394-519 ] of embryo stem cells; 519, 52. 519), the number of clones of total gpscs was counted after 5 days, and the results are shown in fig. 12: the composition (5SM) can obviously improve the number of pluripotent stem cells (5SM-gPSCs) and improve the number by about 1000 times compared with a DMSO control group.
5. Effect of composition (5SM) on the reprogramming time of spermatogonial stem cells
The spermatogonial stem cells were seeded in 24-well plates previously coated with Fibronectin (Fibronectin) in an amount of 4,000 cells per well, the composition (5SM) in 1 was added (control group was prepared by the above composition method, the solvent in an equal amount of concentrate was added to the culture solution of the spermatogonial stem cells at a concentration that was negligible to the toxicity of the spermatogonial stem cells), 6 wells were added to each group, each treatment was repeated 3 times (18 wells for each treatment), 500 μ L of the half solution was replaced every 4 days, and the number of clones of Oct4-GFP positive cells in the wells was counted at 10 days, 13 days, 16 days, and 19 days after the addition of the composition (5SM), as shown in fig. 13: composition (5SM) significantly reduced the reprogramming time of spermatogonial stem cells, with Oct4-GFP positive cells appearing first at day 10, whereas control group had only first few Oct4-GFP positive cells at about day 19.
6. Composition (5SM) induced pluripotent stem cell 5SM-gPSC obtained by reprogramming spermatogonial stem cells, and its ability to differentiate into three germ layers and to chimera into germ lines
(1)5SM-gPSC teratoma formation experiments: the 5SM-gpscs obtained in 3 were collected and injected subcutaneously into the groin of nude mice (7-week-old male mice, purchased from southern medical university laboratory animal care center), and the cells were resuspended in N2B27 liquid at a cell volume of 50 ten thousand per groin side; a tumor was observed to be clearly formed at the injection site 8 weeks after the injection, and after the tumor was removed, the tumor was formalin-fixed and embedded, and then paraffin sections were HE-stained, and the results are shown in fig. 14: 5SM-gPSC can be differentiated in vivo to give a tri-germ layer structure.
(2)5SM-gPSC diploid chimerism experiment: the 5SM-gPSC obtained in Collection 3 was injected into blastocysts. Blastocysts were obtained from 3.5dpc (day postcoitum) ICR dams (purchased from southern university of medical sciences laboratory animal management center) of uterus, 12 SM-gPSCs were injected into each blastocyst cavity by the Japanese Moore micromanipulation system, followed by implantation of 0.5dpc of the oviduct of a pseudopregnant dam, and the results are shown in FIG. 15: oct4-GFP fluorescence was seen in the genital ridges at day 12.5 of the embryo, indicating that 5SM-gPSC has germline chimerism capability.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Claims (10)

  1. Use of an EGFR inhibitor which is at least one of daphnetin and epigallocatechin gallate for increasing the reprogramming efficiency of spermatogonial stem cells.
  2. 2. Use according to claim 1, characterized in that:
    the concentration of the EGFR inhibitor is 2.5-10 mu M.
  3. 3. A composition comprising the following components: EGFR inhibitors, vitamin C, SGC707 and tauroursodeoxycholic acid;
    the EGFR inhibitor is at least one of daphnetin and epigallocatechin gallate.
  4. 4. The composition of claim 3, wherein:
    the composition comprises the following components: daphnetin, epigallocatechin gallate, vitamin C, SGC707, and tauroursodeoxycholic acid.
  5. 5. The composition of claim 4, wherein:
    the composition comprises the following components: 2.5-10 mu M daphnetin, 5-10 mu M epigallocatechin gallate, 283.9-380.4 mu M vitamin C, 2.5-10 mu M SGC707 and 10-20 mu M tauroursodeoxycholic acid.
  6. 6. The composition of claim 5, wherein:
    the composition comprises the following components: 7.5-10 mu M daphnetin, 5-7.5 mu M epigallocatechin gallate, 283.9-380.4 mu M vitamin C, 5-10 mu M SGC707 and 10-20 mu M tauroursodeoxycholic acid.
  7. 7. Use of the composition of any one of claims 3-6 to increase the efficiency of reprogramming of spermatogonial stem cells.
  8. 8. Use of the composition of any one of claims 3 to 6 for shortening the reprogramming time course of spermatogonial stem cells.
  9. 9. A method for improving reprogramming efficiency of spermatogonial stem cells, comprising mixing an EGFR inhibitor or the composition of any one of claims 3 to 6 with spermatogonial stem cells, and culturing;
    the EGFR inhibitor is at least one of daphnetin and epigallocatechin gallate.
  10. 10. A method for shortening the reprogramming time of spermatogonial stem cells, comprising mixing the composition according to any one of claims 3 to 6 with spermatogonial stem cells, and culturing the mixture.
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