CN113481150B - 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 PDFInfo
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Abstract
The invention belongs to the field of stem cell biology and cell reprogramming, and particularly relates to 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, rapidly and efficiently induce the spermatogonial stem cells to reprogram into the pluripotent stem cells with good in-vitro and in-vitro differentiation potential and germ line chimeric ability under the conditions of no introduction and no use of any exogenous gene/transcription factor/MicroRNA (miRNA), related RNA, polypeptide or protein and other induction factors, and the components have good drug properties, low cost, good stability and simple operation.
Description
Technical Field
The invention belongs to the field of stem cell biology and cell reprogramming, and particularly relates to 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 differentiate in multiple directions, i.e., stem cells can produce cells that have the same characteristics as themselves to maintain self-renewal, and can also differentiate to produce functionally specialized cells. The characteristics of stem cells make the stem cells have great application value in the field of regenerative medicine.
Primordial stem cells (spermatogonial stem cells, SSCs) are an adult stem cell that is present in the male testis or testis, and are important guarantees for the maintenance of a continuous spermatogenic process throughout life following puberty. In the long-term in vitro culture process, the mouse spermatogonial stem cells can be spontaneously reprogrammed into pluripotent stem cells (Germ cell derived pluripotent stem cells, gPSCs), and the gPSCs can be amplified and cultured in an embryonic stem cell culture solution, and have good in-vitro and in-vitro differentiation potential and germ line chimeric capability, so that the method has potential application prospect.
However, the previously established spermatogonial stem cell reprogramming system has the defects of low efficiency (about 0.05%), long time (about 28 days), unstable system and the like, so that the research on the mechanism of spontaneous reprogramming of the spermatogonial stem cells and the application thereof are faced with obstacles. Therefore, the development of a system for efficiently and rapidly inducing the reprogramming of spermatogonial stem cells is important for the development of the field of cell reprogramming.
Disclosure of Invention
It is an object of the first aspect of the present invention to provide the use of an EGFR inhibitor for increasing the efficiency of spermatogenic stem cell reprogramming.
The object of the second aspect of the present invention is to provide a composition.
It is an object of a third aspect of the present invention to provide the use of a composition according to the second aspect of the present invention for increasing the efficiency of spermatogonial stem cell reprogramming.
The object of the fourth aspect of the present invention is to provide the use of a composition according to the second aspect of the present invention for increasing the reprogramming time of spermatogonial stem cells.
The fifth aspect of the present invention is directed to a method for improving the reprogramming efficiency of spermatogonial stem cells.
The sixth aspect of the present invention is directed to a method for shortening spermatogonial stem cell reprogramming Cheng Shicheng.
In order to achieve the above 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 spermatogenic stem cell reprogramming.
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 to 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.
Still further preferred, 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 to improve the efficiency of spermatogonial stem cell reprogramming.
In a fourth aspect of the invention there is provided the use of a composition of the second aspect of the invention for shortening spermatogonial stem cell reprogramming Cheng Shicheng.
In a fifth aspect of the invention, there is provided a method of increasing the reprogramming efficiency of spermatogonial stem cells by mixing an EGFR inhibitor or a composition according to the third aspect of the 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 to 10. Mu.M.
In a sixth aspect of the invention, there is provided a method of shortening spermatogonial stem cell reprogramming Cheng Shicheng, mixing a composition of the third aspect of the invention with spermatogonial stem cells, and culturing.
The beneficial effects of the invention are as follows:
the invention discloses application of EGFR inhibitor (daphnetin and/or epigallocatechin gallate) in improving the reprogramming efficiency of spermatogonial stem cells for the first time, which can obviously improve the reprogramming efficiency of 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 obviously improve the reprogramming Cheng Xiaolv of the spermatogonial stem cells (about 100 times of the composition), the clone number of the Oct4-GFP positive cells after the composition treats the spermatogonial stem cells is larger than the sum of the clone numbers of the Oct4-GFP positive cells after the composition treats the spermatogonial stem cells, namely the reprogramming efficiency of the composition on the spermatogonial stem cells is larger than the sum of the reprogramming efficiency of the composition on the spermatogonial stem cells, which indicates that the synergistic effect can be achieved among the components of the composition; meanwhile, the composition can increase the number of the obtained pluripotent stem cells (about 1000 times of improvement), shorten the reprogramming Cheng Shicheng of the spermatogonial stem cells (about 1 time of acceleration and 10 days), and induce the pluripotent stem cells obtained by reprogramming of the spermatogonial stem cells to have 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 in-vitro and in-vivo differentiation potential and germ line chimeric capability under the condition of not introducing and using any exogenous gene/transcription factor/MicroRNA (miRNA), related RNA, polypeptide or protein and other induction factors, and the components have good drug property, low cost, good stability and simple operation.
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FIG. 1 is a bright field diagram of spermatogonial stem cells of example 1.
FIG. 2 is a statistical plot of the number of Oct4-GFP positive cell clones obtained by reprogramming spermatogonial stem cells at different concentrations of daphnetin in example 1: * Represents p <0.05.
FIG. 3 is a statistical chart of the number of Oct4-GFP positive cell clones obtained by reprogramming spermatogonial stem cells at different concentrations of epigallocatechin gallate in example 1: * Represents p <0.05.
FIG. 4 is a statistical plot of the number of Oct4-GFP positive cell clones obtained from the reprogramming of spermatogonial stem cells at different concentrations of vitamin C in example 1: * Represents p <0.05.
FIG. 5 is a statistical plot of the number of Oct4-GFP positive cell clones obtained from the reprogramming of spermatogonial stem cells at various concentrations of SGC707 in example 1: * Represents p <0.05.
FIG. 6 is a statistical plot of the number of Oct4-GFP positive cell clones obtained by reprogramming spermatogonial stem cells at different concentrations of tauroursodeoxycholic acid in example 1: * Represents p <0.05.
FIG. 7 is a statistical chart of the number of Oct4-GFP positive clones obtained by reprogramming spermatogonial stem cells with vitamin C and tauroursodeoxycholic acid at various concentrations in example 2: * Represents p <0.05.
FIG. 8 is a statistical chart of the number of Oct4-GFP positive clones obtained by reprogramming spermatogonial stem cells in example 2 with vitamin C, tauroursodeoxycholic acid and daphnetin at various concentrations: * Represents p <0.05.
FIG. 9 is a statistical chart of the number of Oct4-GFP positive clones obtained by reprogramming spermatogonial stem cells in example 2 under SGC707 of vitamin C, tauroursodeoxycholic acid, daphnetin and different concentrations: * Represents p <0.01.
FIG. 10 is a statistical plot of the number of Oct4-GFP positive clones obtained by reprogramming spermatogonial stem cells in example 2 with vitamin C, tauroursodeoxycholic acid, daphnetin, SGC707, and varying concentrations of epigallocatechin gallate: * Represents p <0.05.
FIG. 11 is a graph showing the effect of the composition of example 2 on the reprogramming efficiency of spermatogonial stem cells: wherein A is Oct4-GFP positive cell clone obtained by reprogramming spermatogonial stem cells after composition treatment, and an alkaline phosphatase staining positive clone detection result diagram; b is a statistical chart of the clone numbers of Oct4-GFP positive cells obtained by reprogramming spermatogonial stem cells after the composition treatment: * P <0.0001.
FIG. 12 is a graph showing the effect of the composition of example 2 on the number of pluripotent stem cells obtained by reprogramming spermatogenic stem cells: wherein A is an intuitive graph of the number of pluripotent stem cells obtained by reprogramming spermatogonial stem cells after treatment with the composition; b is a statistical chart of the number of the pluripotent stem cells obtained by reprogramming the spermatogonial stem cells after the composition treatment: * P <0.0001.
FIG. 13 is a graph showing the effect of the composition of example 2 on the reprogramming time of spermatogonial stem cells.
FIG. 14 is a diagram showing the three germ layers differentiation of pluripotent stem cells obtained by reprogramming spermatogonial stem cells induced by the composition of example 2.
FIG. 15 is a graph showing the results of functional verification of pluripotent stem cells obtained by inducing reprogramming of spermatogonial stem cells with the composition of example 2: wherein A is the bright field and fluorescence image of the embryo reproduction ridge 12 days after 5SM-gPSC blastula injection; b is a picture of F0 generation chimeric mice born after 5SM-gPSC blastula injection.
Detailed Description
The present invention will be described in further detail with reference to specific examples.
It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention.
The experimental methods, in which specific conditions are not noted in the following examples, are generally conducted under conventional conditions or under conditions recommended by the manufacturer. The materials, reagents and the like used in this example are commercially available ones unless otherwise specified.
Example 1 Effect of Daphnetin (Daphnetin), epigallocatechin gallate ((-) -Epigallocatechin Gallate, EGCG), vitamin C (Vc), SGC707, tauroursodeoxycholic acid (TUDCA) on the efficiency of spermatogenic Stem cell reprogramming
1. Establishment of spermatogonial stem cell line
1 mouse (C57 BL/6x DBA) was taken 5.5 days after birth, and the mouse was sacrificed by cervical dislocation, and the abdominal skin of the mouse was cut off. Pulling out testis, placing in a 3.5cm cell culture dish containing PBS, and removing testis white membrane in the dish; transferring the washed testis seminiferous tubules to a new 3.5cm cell culture dish filled with PBS, adding collagenase IV, digesting for 7 minutes, observing most of the tubular cavities under a lens, adding 2mL of pancreatin containing 0.05% EDTA, digesting for 15 minutes to a single cell state, stopping digestion by using DMEM containing 10% fetal calf serum, filtering by using a 70 μm cell sieve, and re-suspending by using a seminoma stem cell culture solution (specific formula reference: kanatsu-Shinohara M.Long-Term Proliferation in Culture and Germline Transmission of Mouse Male Germline Stem Cells [ J ]. BIOLOGY OF REPRODUCTION,2003.69:5 ], planting on a cell culture plate coated with gelatin in advance, and after 24 hours, observing that the testis body cell part is adhered to the bottom of the gelatin-prepared culture plate due to difference, and transferring the culture solution to a new 12-hole culture plate while most of germ cells in testis are still in a suspended state, wherein half of the liquid needs to be replaced every other day. After the primary culture was continued for 7 days, the spermatogonial stem cells were seen to be grape-like clones by microscopic observation (FIG. 1), growing on top of somatic cells. At this time, the cells can be passaged by pancreatin digestion, wherein the passaged cells are digested with 0.05% pancreatin at 37 ℃ for 2min, and the digestion is stopped with DMEM containing 10% fetal bovine serum after the single cells are digested, so as to obtain spermatogenic stem cells.
2. Configuration of small molecular compound concentration storage
Respectively dissolving Daphnetin (Daphnetin), epigallocatechin gallate ((-) -Epigallocatechin Gallate, EGCG), SGC707, tauroursodeoxycholic acid (TUDCA) in DMSO, and vitamin C in water; under aseptic conditions, all five small molecules above were configured for 100mM storage. For example: daphnetin dissolved 17.81mg of the powder in 1mL of DMSO to give a final concentration of 100 mM; ECGC dissolved 45.84mg of the powder in 1mL of DMSO to give a final concentration of 100 mM; SGC707 dissolved 29.83mg of the powder in 1mL of DMSO to give a final concentration of 100 mM; TUDCA 49.97mg of the powder was dissolved in 1mL of DMSO to give a final concentration of 100 mM; vitamin C17.6 mg of the powder was dissolved in 1mL of water to give a final concentration of 100 mM. After the compound is completely dissolved, split charging is carried out, the split charging is preserved in a refrigerator at the temperature of minus 20 ℃, and when the split charging type SSC reprogramming is used, the compound is concentrated, and the culture solution of the spermatogonial stem cells is added and diluted to a specific concentration.
3. Effect of daphnetin on spermatogonial Stem cell reprogramming efficiency
Sperm stem cells were seeded into 24-well dishes pre-packed with Fibronectin (Fibronectin) at a cell amount of 4,000 sperm stem cells, daphnetin (final concentration 0, 2.5, 5, 7.5, 10 μm, respectively, as the solvent in the sperm stem cell culture solution) was added, each treatment was repeated 3 times (6 wells per group, 18 wells per treatment total), 500 μl per well, half-liquid was replaced every 4 days, and after 19 days of culture, the number of Oct4-GFP positive cell clones per well (as indicated by the occurrence of reprogramming success of Oct4-GFP strong positive clones) was examined, as shown in fig. 2: daphnetin with the concentration of 2.5-10 mu M can obviously improve the reprogramming efficiency of spermatogonial stem cells, and especially has the best reprogramming effect when the concentration is 7.5 mu M.
4. Effect of epigallocatechin gallate ((-) -Epigallocatechin Gallate, EGCG) on spermatogonial stem cell reprogramming efficiency
Sperm stem cells were seeded into 24-well dishes pre-packed with Fibronectin (Fibronectin) at a cell amount of 4,000 sperm stem cells, EGCG (final concentration 0, 2.5, 5, 7.5, 10 μm, respectively, as the solvent in sperm stem cell culture) was added, each treatment was repeated 3 times (6 wells per group, 18 wells per treatment, 500 μl per well, half-liquid was replaced every 4 days, and after culturing for 19 days, the number of Oct4-GFP positive cell clones per well (as an indicator of reprogramming success by the appearance of Oct4-GFP strong clones) was examined, and the results are shown in fig. 3: when the concentration is 5-10 mu M, EGCG can obviously improve the reprogramming efficiency of spermatogonial stem cells.
5. Effect of vitamin C (Vc) on the efficiency of spermatogonial stem cell reprogramming
Sperm stem cells were seeded into 24-well dishes pre-packed with Fibronectin (Fibronectin) at a cell amount of 4,000 sperm stem cells, vc (final concentration 0, 113.6, 170.3, 283.9, 380.4 μm, respectively, solvent in sperm stem cell culture broth) was added, each treatment was repeated 3 times (6 wells per group, 18 wells per treatment total), 500 μl per well, half-liquid was replaced every 4 days, and after 19 days of culture, the number of Oct4-GFP positive cell clones per well (as indicated by the occurrence of reprogramming success of Oct4-GFP strong positive clones) was examined, as shown in fig. 4: when the concentration is 283.9-380.4 mu M, vc can obviously improve the reprogramming efficiency of spermatogonial stem cells.
Effect of SGC707 on spermatogonial Stem cell reprogramming efficiency
Sperm stem cells were seeded into 24-well dishes pre-packed with Fibronectin (Fibronectin) at a cell amount of 4,000 sperm stem cells, SGC707 (final concentration 0, 2.5, 5, 7.5, 10 μm, respectively, as the solvent in the sperm stem cell culture solution) was added, each treatment was repeated 3 times (6 wells per group, 18 wells per treatment total), 500 μl per well, half-liquid was replaced every 4 days, and after culturing for 19 days, the number of Oct4-GFP positive cell clones per well (as indicated by the occurrence of reprogramming success of Oct4-GFP strong positive clones) was examined, and the results are shown in fig. 5: SGC707 may significantly increase the efficiency of spermatogonial stem cell reprogramming when the concentration is 2.5-10. Mu.M.
7. Effect of tauroursodeoxycholic acid (TUDCA) on the efficiency of spermatogonial stem cell reprogramming
Sperm stem cells were seeded into 24-well dishes pre-packed with Fibronectin (Fibronectin) at a cell amount of 4,000 sperm stem cells, TUDCA (final concentration 0, 2.5, 5, 10, 20. Mu.M, respectively, as the solvent in the sperm stem cell culture solution) was added, each treatment was repeated 3 times (6 wells per group, 18 wells per treatment, 500. Mu.L per well, half-liquid was changed every 4 days, and after 19 days of culture, the number of Oct4-GFP positive cell clones per well (as an indicator of reprogramming success by the appearance of Oct4-GFP strong clones) was examined, and the results are shown in FIG. 6: SGC707 may significantly increase the efficiency of spermatogonial stem cell reprogramming at concentrations of 10-20. Mu.M.
Example 2A composition
1. Determination of the concentration of the Components (daphnetin, epigallocatechin gallate, vitamin C, SGC707 and Tauroursodeoxycholic acid) in composition (5 SM)
After determining that the optimal concentration of vitamin C (Vc) for promoting spermatogonial stem cells is 380.4 mu M, the next small molecules are added one by one on the basis, and the optimal combined concentration is tested: the spermatogonial stem cells were seeded into 24-well dishes pre-packed with Fibronectin (Fibronectin) at a cell amount of 4,000 spermatogonial stem cells per well, and the compositions Vc (final concentration 380.4. Mu.M) +TUDCA were added at 5.0. Mu.M, 10. Mu.M, 20. Mu.M, respectively, with the control group only Vc (concentration 380.4. Mu.M) added. Each treatment was repeated 3 times (6 duplicate wells per group, 18 duplicate wells per treatment), 500 μl per well, half-liquid was changed every 4 days, and after 19 days of culture, the number of Oct4-GFP positive cell clones per well (as an indicator of the success of reprogramming with the appearance of Oct4-GFP strong positive clones) was examined and the results are shown in fig. 7: when Vc concentration is 380.4 mu M and TUDCA concentration is 10 mu M, the reprogramming efficiency of spermatogonial stem cells can be remarkably improved.
After determining the concentration of Vc+TUDCA, daphnetin addition concentration was tested on this basis. The spermatogonial stem cells were seeded into 24-well dishes pre-packed with Fibronectin (Fibronectin) at a cell amount of 4,000 spermatogonial stem cells, and the compositions Vc (final concentration 380.4. Mu.M) +TUDCA (final concentration 10. Mu.M) +Daphnetin, were added, respectively, at final concentrations of Daphnetin 5.0. Mu.M, 7.5. Mu.M, 10. Mu.M, respectively, at which time the control group was added with Vc alone (final concentration 380.4. Mu.M) +TUDCA (final concentration 10. Mu.M). Each treatment was repeated 3 times (6 replicate wells per group, 18 replicate wells per treatment total), the amount of fluid per well was 500 μl, the half fluid was changed every 4 days, and after 19 days of culture, the number of Oct4-GFP positive cell clones per well (as an indicator of the success of reprogramming with the occurrence of Oct4-GFP strong positive clones) was examined and the results are shown in fig. 8: when the Vc concentration is 380.4 mu M and the TUDCA concentration is 10 mu M, the Daphnetin concentration is 7.5 mu M, so that the reprogramming efficiency of spermatogonial stem cells can be remarkably improved.
After determining the concentration of Vc+TUDCA+Daphnetin, the SGC707 concentration was tested on this basis. The spermatogonial stem cells were inoculated into 24-well dishes pre-packed with Fibronectin (Fibronectin) at a cell amount of 4,000 spermatogonial stem cells, and the compositions Vc (final concentration 380.4. Mu.M) +TUDCA (final concentration 10. Mu.M) +Daphnetin) +SGC707 were added, respectively, at final concentrations of 5.0. Mu.M, 7.5. Mu.M, 10. Mu.M, respectively, to the control group, at which time Vc (final concentration 380.4. Mu.M) +TUDCA (final concentration 10. Mu.M) +Daphnetin (final concentration 10. Mu.M) was added, respectively. Each treatment was repeated 3 times (6 replicate wells per group, 18 replicate wells per treatment total), the amount of fluid per well was 500 μl, the half fluid was changed every 4 days, and after 19 days of culture, the number of Oct4-GFP positive cell clones per well (as an indicator of the success of reprogramming with the occurrence of Oct4-GFP strong positive clones) was examined and the results are shown in fig. 9: when the Vc concentration is 380.4 mu M, the TUDCA concentration is 10 mu M, and the Daphnetin concentration is 7.5 mu M, the SGC707 concentration is 7.5 mu M, so that the reprogramming efficiency of spermatogonial stem cells can be remarkably improved.
After determining the concentration of Vc+TUDCA+Daphnetin+SGC707, the concentration of (-) -Epigallocatechin Gallate was tested on this basis. The spermatogonial stem cells were inoculated into 24-well dishes pre-packed with Fibronectin (Fibronectin) at a cell amount of 4,000 spermatogonial stem cells, and the compositions Vc (final concentration: 380.4. Mu.M) +TUDCA (final concentration: 10. Mu.M) +Daphnetin (final concentration: 10. Mu.M) +SGC707 (final concentration: 7.5. Mu.M) +(-) -Epigallocatechin Gallate, (-) -Epigallocatechin Gallate final concentrations: 5.0. Mu.M, 7.5. Mu.M, 10. Mu.M, respectively, were added to the control group, at which time Vc (final concentration: 380.4. Mu.M) +TUDCA (final concentration: 10. Mu.M) +Daphnetin (final concentration: 10. Mu.M) +SGC707 (final concentration: 7.5. Mu.M) were added. Each treatment was repeated 3 times (6 replicate wells per group, 18 replicate wells per treatment total), the amount of fluid per well was 500 μl, the half fluid was changed every 4 days, and after 19 days of culture, the number of Oct4-GFP positive cell clones per well (as an indicator of the success of reprogramming with the occurrence of Oct4-GFP strong positive clones) was examined and the results are shown in fig. 10: when Vc concentration is 380.4 mu M, TUDCA concentration is 10 mu M, daphnetin concentration is 7.5 mu M, SGC707 concentration is 7.5 mu M, (-) -Epigallocatechin Gallate concentration is 7.5 mu M, and reprogramming efficiency of spermatogonial stem cells can be remarkably improved.
2. A composition (5 SM) consisting of: daphnetin (Daphnetin) 7.5 μM, epigallocatechin gallate ((-) -Epigallocatechin Gallate, EGCG) 7.5 μM, vitamin C (Vc) 67 μg/mL, SGC7077.5 μM and Tauroursodeoxycholate (TUDCA) 10 μM; the preparation method comprises the following steps: the stock solutions of the compounds of example 1 were used and added to the spermatogonial stem cell culture medium, respectively, to obtain the extract.
3. Effect of composition (5 SM) on spermatogonial stem cell reprogramming efficiency
The sperm stem cells were inoculated into 24-well dishes pre-packed with Fibronectin (Fibronectin) at a cell amount of 4,000 sperm stem cells per well, and the composition (5 SM) of 1 was added separately (control group, the solvent in the equal amount of concentrate was added to the sperm stem cell culture solution according to the composition preparation method described above), 6 wells per group were repeated 3 times per treatment (18 wells per treatment total), 500 μl per well, half-liquid was replaced every 4 days, and after 19 days of culture, the number of Oct4-GFP positive cell clones per well (as an indication of the occurrence of reprogramming success of Oct4-GFP strong positive clones) and alkaline phosphatase (Alkaline phosphatase, AP) staining positive clones were examined, and the results are shown in fig. 11: composition (5 SM) can significantly improve the reprogramming efficiency of spermatogonial stem cells, wherein the number of Oct4-GFP positive cell clones in composition (5 SM) (about 90) is about 100 times that of the control group (DMSO) and greater than the sum of the number of Oct4-GFP positive cell clones in each component (4+9+13+10+12=48), indicating that each component in composition (5 SM) produces a synergistic effect.
4. Effect of composition (5 SM) on the number of acquired pluripotent Stem cells (Germ cell derived pluripotent stem cells, gPSCs)
The preparation method comprises the steps of inoculating spermatogonial stem cells into 24-well dishes pre-packed with Fibronectin (Fibronectin) at a cell amount of 4,000 spermatogonial stem cells, adding the composition (5 SM) in 1 to the dishes (control group, according to the preparation method of the composition, taking the solvent in an equivalent concentrate to add the spermatogonial stem cell culture fluid, the concentration of which is negligible for the toxicity of the spermatogonial stem cells), repeating 3 times per group of 6 duplicate wells (18 duplicate wells per treatment), replacing half-liquid per 500 μl per well for 4 days, and after culturing for 19 days, digesting all cells in the wells to embryonic stem cell culture fluid containing N2B27+CHIR99021+PD 03259501+mLIF2i (specific formulation reference: YIng QL, wray J, nichols J, et al. The ground state of embryonic stem cell self-renewal [ J ] Nature5725 (7194) PSC, 2008.453) and total cloning count after 5 days is shown in FIG. 12: the composition (5 SM) can significantly increase the number of the pluripotent stem cells (5 SM-gPSCs) by about 1000 times compared with a DMSO control group.
5. Effect of composition (5 SM) on the Probiotics reprogramming time course
The method comprises the steps of inoculating the spermatogonial stem cells into 24-well culture dishes pre-packed with Fibronectin (Fibronectin) at a cell amount of 4,000 spermatogonial stem cells, adding the composition (5 SM) in 1 to the dishes (control group, according to the preparation method of the composition, taking the solvent in an equivalent concentrated reservoir, adding the spermatogonial stem cell culture solution, the concentration of which is negligible to the toxicity of the spermatogonial stem cells), repeating 3 times per group of 6 duplicate wells (18 duplicate wells per treatment), replacing half liquid every 4 days with 500 mu L of each well, and counting the clone numbers of Oct4-GFP positive cells in the wells on the 10 th day, 13 th day, 16 th day and 19 th day after adding the composition (5 SM), wherein the results are shown in FIG. 13: composition (5 SM) can significantly shorten the time course of spermatogonial stem cell reprogramming, with Oct4-GFP positive cells occurring earliest to day 10, whereas the control group had a small number of Oct4-GFP positive cells occurring only initially about day 19.
6. Composition (5 SM) induces the tricdermal differentiation and germ line chimeric ability of pluripotent stem cells 5SM-gPSC obtained by reprogramming spermatogenic stem cells
(1) 5SM-gPSC teratoma formation experiments: 5SM-gPSC obtained in 3 was collected for inguinal subcutaneous injection in nude mice (7 Zhou Lingxiong mice, purchased from the experimental animal management center of southern medical university), and cells were resuspended in N2B27 solution at 50 ten thousand cells per inguinal side; obvious tumor formation at the injection site was observed 8 weeks after injection, and after taking out the tumor, the tumor was formalin-fixed and embedded, and then paraffin section HE staining was performed, and the results are shown in fig. 14: the 5SM-gPSC can differentiate in vivo to obtain a three-germ layer structure.
(2) 5SM-gPSC diploid chimeric experiments: the 5SM-gPSC obtained in 3 was collected for blastula injection. Blastocysts were taken from the uterus of 3.5dpc (day postcoitum) ICR mice (purchased from the Experimental animal management center of the university of south medical science), 12 5SM-gPSCs were injected into each blastocyst chamber by the Japanese metallocene micromanipulation system, and then the fallopian tubes of the 0.5dpc pseudopregnant mice were transplanted, and the results are shown in FIG. 15: the genital ridge was seen to have Oct4-GFP fluorescence at day 12.5 of the embryo, indicating that 5SM-gPSC had germ line chimeric ability.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Claims (6)
1. A composition characterized by:
the composition consists of the following components: 7.5 mu M daphnetin, 5-10 mu M epigallocatechin gallate, 380.4 mu M vitamin C, 7.5-10 mu MSGC707 and 10 mu M tauroursodeoxycholic acid.
2. The composition of claim 1, wherein:
the concentration of the epigallocatechin gallate is 5-7.5 mu M.
3. Use of a composition according to any one of claims 1-2 for increasing the efficiency of spermatogonial stem cell reprogramming in vitro.
4. Use of a composition according to any one of claims 1-2 for in vitro shortening of spermatogonial stem cell reprogramming Cheng Shicheng.
5. A method for improving the reprogramming efficiency of spermatogonial stem cells in vitro, mixing the composition of any one of claims 1-2 with spermatogonial stem cells, and culturing.
6. A method of shortening spermatogonial stem cells in vitro by mixing and culturing the composition of any one of claims 1-2 with spermatogonial stem cells.
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