CN111269940A

CN111269940A - Method for directly transdifferentiating mesenchymal stem cells into sperms by using transcription factor FOXO1

Info

Publication number: CN111269940A
Application number: CN202010104321.1A
Authority: CN
Inventors: 马廉
Original assignee: Shenzhen Childrens Hospital
Current assignee: Shenzhen Childrens Hospital
Priority date: 2020-02-20
Filing date: 2020-02-20
Publication date: 2020-06-12

Abstract

The invention provides a method for directly transdifferentiating mesenchymal stem cells into sperms by using a transcription factor FOXO1, which comprises the following specific steps: the transcription factor FOXO1 is introduced into the mesenchymal stem cells by means of transfection, and then the mesenchymal stem cells are induced and cultured into the sperms. The method introduces the transcription factor FOXO1 into the mesenchymal stem cells to induce the mesenchymal stem cells to develop towards sperms, is not only helpful for understanding the regulation mechanism of the stem cells to the organism life activities, discussing the signal path for maintaining the SSCs dynamic balance of the spermatogonial stem cells, but also brings a new scheme for inheriting the own biological information for male sterility, especially for the treatment of male azoospermia patients.

Description

Method for directly transdifferentiating mesenchymal stem cells into sperms by using transcription factor FOXO1

Technical Field

The invention relates to the technical field of biology, in particular to a method for directly transdifferentiating mesenchymal stem cells into sperms by using a transcription factor FOXO 1.

Background

Infertility is a global medical and social problem, and about 10% -20% of all the sexy couples suffer from infertility, wherein male factors account for about 50%, mainly manifested as azoospermia and sperm motility insufficiency.

For the infertile patients with spermatogenesis deficiency, the existing treatment means such as medicines, operations, auxiliary reproductive technologies and the like still cannot solve the fertility problem of the infertile patients. Therefore, the research on the regulation and control mechanism of related molecules in the process of spermatogenesis development and the search for the mesenchymal stem cells from the patient to develop the mesenchymal stem cells into functional germ cells for the accurate treatment of male infertility are the research hotspots of the male infertility treatment at present.

The results of the existing studies have demonstrated that: the mesenchymal stem cells have the capacity of differentiating into male germ cells, and the mesenchymal stem cells can be promoted to transdifferentiate towards SSCs by various different culture conditions or induction schemes at present.

Mesenchymal stem cells are induced by retinoic acid, co-cultured with testis supporting cells or transplanted into seminiferous tubules of sterile mice to express early markers of male germ cells, but can not start meiosis, the mesenchymal stem cells are reprogrammed to Induced Pluripotent Stem Cells (iPSCs), after being induced by BMP4, markers related to meiosis can be expressed, but cell differentiation efficiency is low, downstream gene expression cannot be detected, mature sperms are not obtained, the mesenchymal stem cells cultured in a testis cell condition culture medium containing retinoic acid and testosterone are morphologically changed, and germ cell markers such as Oct-4, α integrin, Stella, C-kit and VASA are expressed, human umbilical cord mesenchymal stem cells, human amniotic epithelial cells and chorion plate cells are cultured together, and a small number of mesenchymal stem cell-like cells in a shape like by induction culture, such as bone morphogenetic protein 4(BMP4) and Retinoic Acid (RA) are added to obtain a small number of mesenchymal stem cell-like cells cultured in a manner of inducing culture, and the mesenchymal stem cells cultured with pollakin cells are cultured by using a PCR-EB (2) to obtain round stem cells, and the cells cultured in vitro, and the like⁺The cells are paved on a feeder layer of mouse embryo fibroblasts and are subjected to induction culture with BMP4, the cell morphology is changed into a sperm-like state, male germ cell markers PLZF, GFRa1, DMRT1 and the like are expressed, and immunofluorescence shows that the apical voxel Acrosin is positive.

In summary, in the existing culture conditions or induction schemes for inducing mesenchymal stem cells to transdifferentiate towards SSCs, the detectable markers are only early markers of male germ cells, no markers of the cells entering the meiotic stage are detected, and mature and functional male germ cells are not obtained.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a method for directly transdifferentiating mesenchymal stem cells into sperms by using a transcription factor FOXO 1.

In order to achieve the purpose, the invention adopts the following technical scheme:

the invention provides a method for directly transdifferentiating mesenchymal stem cells into sperms by using a transcription factor FOXO1, which comprises the following specific steps: the transcription factor FOXO1 is introduced into mesenchymal stem cells by means of transfection, and then the mesenchymal stem cells are cultured into sperms.

Further, the transcription factor FOXO1 is introduced into the mesenchymal stem cells by means of lentivirus transfection, and the method comprises the following steps:

step one, calculating the addition amount of a lentivirus carrying FOXO1 gene added into each hole, then adding the lentivirus into a culture medium containing mesenchymal stem cells, and adding a transfection accelerating agent for transfection;

and step two, after the transfection is carried out for a period of time and the cells are well recovered, removing the original culture medium, and carrying out screening culture for a period of time by using a screening culture medium to obtain a stable cell strain.

Wherein, the preparation method of the complete culture medium comprises the following steps: the basal medium was DMEM/F-12 supplemented with 10% FBS prior to use. The preparation method of the screening culture medium comprises the following steps: the basal medium was DMEM/F-12 supplemented with 10% FBS, 200ug/mL hygromycin B prior to use.

Further preferably, the transfection accelerating agent in the first step is polybrene.

Further preferably, the screening medium in step two is a hygromycin B-containing medium.

Further, the transcription factor FOXO1 is introduced into the mesenchymal stem cells by electrotransfection, and the method comprises the following steps:

firstly, selecting mesenchymal stem cells in logarithmic growth phase, digesting, washing, resuspending and counting, mixing with a plasmid carrying FOXO1 gene, and adding into an electric transfer cup;

and step two, placing the electric rotating cup on the electric rotating cup frame, and setting electric transfection parameters for transfection.

Further, the transcription factor FOXO1 is introduced into the mesenchymal stem cells by means of lipofection, and comprises the following steps:

fully mixing a mixed solution A containing liposome and a mixed solution B containing FOXO1 gene plasmid to form a transfection mixed solution containing a DNA-liposome compound;

adding the mesenchymal stem cells into the transfection mixed solution, and culturing for 7-9h in a cell culture box;

and step three, after the culture, replacing a mesenchymal stem cell culture medium containing FBS, and then placing the mesenchymal stem cell culture medium in a cell culture box for continuous incubation until the transfection is finished.

Further preferably, in the first step, the mixed solution A also comprises an Opti-MEM culture medium.

Further preferably, in the first step, the mixed solution B also comprises an Opti-MEM culture medium and an enhancer.

By adopting the technical scheme, compared with the prior art, the invention has the following technical effects:

the method for directly transdifferentiating the mesenchymal stem cells into sperms by using the transcription factor FOXO1, provided by the invention, introduces the transcription factor FOXO1 into the mesenchymal stem cells to induce the development of the mesenchymal stem cells towards sperms, is not only beneficial to understanding the regulation and control mechanism of the stem cells on the life activities of organisms, discussing a signal path for maintaining the dynamic balance of the spermatogonial stem cells SSCs, but also brings a new scheme for inheriting own biological information for male sterility, particularly for treating male azoospermia patients.

Drawings

Fig. 1 is a graph showing the morphological results of mesenchymal stem cells overexpressing FOXO1 in a validation experiment according to the present invention;

FIG. 2 is a diagram showing the statistical results of the expression levels of the primordial germ cell-related marker genes in a validation experiment according to the present invention;

FIG. 3 is a diagram showing the statistical results of the expression levels of the related marker genes of spermatogonial stem cells in a validation experiment according to the present invention;

FIG. 4 is a graph showing the statistical results of the expression levels of meiosis-associated marker genes in a validation experiment according to the present invention;

FIG. 5 is a graph showing the statistical results of expression levels of sperm related marker genes in a validation experiment according to the present invention;

FIG. 6 is a FISH result graph in a verification experiment according to the present invention;

FIG. 7 shows the result of RT-PCR assay of Primordial Germ Cell (PGC) -specific markers OCT4, SOX17 in a validation experiment of the present invention;

FIG. 8 shows the detection results of RT-PCR of specific markers UCHL1 and ID4 of Spermatogonial Stem Cells (SSCs) in a validation experiment according to the present invention;

FIG. 9 shows the RT-PCR detection results of meiotic markers KIT and STRA8 in a validation experiment according to the present invention;

FIG. 10 shows the results of RT-PCR assay of sperm cell markers PRM1 and TNP2 in a validation experiment in accordance with the present invention;

FIG. 11 is a graph showing the results of electrophoresis of the expression of the specific markers OCT4 and SOX17 protein for Primordial Germ Cells (PGC) in a validation experiment according to the present invention;

FIG. 12 is a graph showing the results of electrophoresis of the expression of the specific markers UCHL1 and ID4 protein of Spermatogonial Stem Cells (SSCs) in a validation experiment according to the present invention;

FIG. 13 is a graph showing the results of electrophoresis of the expression of meiotic marker KIT and STRA8 proteins in a validation experiment according to the present invention;

FIG. 14 is a graph showing the results of electrophoresis of the expression of the sperm cell marker TNP2 protein in a validation experiment in accordance with the present invention.

Detailed Description

The invention provides a method for directly transdifferentiating mesenchymal stem cells into sperms by using a transcription factor FOXO1, which comprises the following specific steps: the transcription factor FOXO1 is introduced into the mesenchymal stem cells by means of transfection, and then the mesenchymal stem cells are induced and cultured into the sperms.

In a preferred embodiment, the transcription factor FOXO1 is introduced into the mesenchymal stem cells by means of lentivirus transfection, and comprises the following steps:

step one, calculating the addition amount of a lentivirus carrying FOXO1 gene added into each hole, then adding the lentivirus into a culture medium containing mesenchymal stem cells, and then adding polybrene for transfection;

and step two, after the transfection is carried out for a period of time and the cells are well recovered, removing the original culture medium, and selecting and culturing for a period of time by using a culture medium containing hygromycin B to obtain a stable cell strain.

In a preferred embodiment, the transcription factor FOXO1 is introduced into mesenchymal stem cells by electrotransfection, comprising the following steps:

In a preferred embodiment, the transcription factor FOXO1 is introduced into mesenchymal stem cells by lipofection, comprising the following steps:

In a preferred embodiment, mixture A in step one further comprises Opti-MEM medium; in the first step, the mixed solution B also comprises Opti-MEM culture medium and an enhancer.

The present invention will be described in detail and specifically with reference to the following examples to facilitate better understanding of the present invention, but the following examples do not limit the scope of the present invention.

Example 1

This example provides a method for introducing the transcription factor FOXO1 into the mesenchymal stem cells by means of lentivirus transfection, comprising the following steps:

green fluorescent control lentiviruses (rLV-ZsGreen control lentiviruses) are selected to infect the HuMSCs, the infection efficiency of cells is observed, and the optimum infection conditions are searched.

(1) Preparing the cells of interest

1) HuMSCs that have proliferated to 80% -90% in a T75 flask were digested with 0.05% pancreatin, and when the cells were observed under a microscope to shrink rapidly to a round shape, complete medium containing 10% FBS was immediately added to terminate the digestion, and the cells were collected into a 15mL centrifuge tube.

2) Centrifuging at 1000rpm for 5min, removing supernatant, adding fresh complete culture medium to gently blow off cells, counting with a hemocytometer, and making into 3-5 × 10⁴cells/mL cell suspension is inoculated into a culture plate for continuous culture, and the plating amount reaches about 30% when infection occurs.

(2) Lentiviral infection of target cells

1) Calculating the lentivirus dosage according to the formula: amount of virus added per well (μ l) × MOI value × cell number/virus stock titer (TU/ml) × 10³。

2) The cells before infection are ensured to be in good state, ZsGreen control lentiviruses with no lentivirus and MOI values of 20, 40, 60, 80 and 100 are used for transfecting HuMSCs after about 20 hours, 2-3 multiple wells are arranged in each group, and 1mg/mL polybrene transfection accelerating agent is added to ensure that the final concentration of each well is 5 mug/mL.

3) After 24h of infection, the stock culture was discarded, fresh medium was added and the transfection effect was observed under a fluorescent microscope.

Construction of FOXO1 overexpressing HuMSCs

(1) And infecting the target cells under the optimum infection condition by the same steps as the previous steps.

(2) After 48h of cell infection, the cells recovered well, the original culture medium was removed, the cells were screened with hygromycin B-containing medium, washed once with DPBS, and screened by adding hygromycin B screening medium to a final concentration of 200 ug/mL.

(3) Observing the cells every day, comparing the growth conditions of the cells of the virus transfection group and the control group, replacing the screening culture medium (200ug/mL Hygromycin B) every 2-3 days, if the cell state is seriously affected or a large number of cells die, reducing the concentration of the Hygromycin B to 50-100ug/mL to slightly recover the cell state, then changing to increase the screening concentration for screening, and repeating the screening for about 7-10 days.

(4) And (5) freezing and storing the stable cell strain.

Example 2

This example provides a method for introducing the transcription factor FOXO1 into the mesenchymal stem cells by means of electrotransfection, comprising the following steps:

(1) selecting 3-5 generations of HuMSCs to ensure that the HuMSCs are in a logarithmic growth phase (because cells in the logarithmic growth phase are vigorous in division and poor in surface structure compactness, exogenous DNA can more easily enter cells in a mitotic phase after the cells are subjected to electrotransfection), digesting the cells by using trypsin, and collecting the cells after the digestion is stopped; centrifuging for 5 minutes at 1000g, discarding the supernatant, adding EP buffer, after resuspending the cells, continuing centrifuging, washing for 2-3 times with the EP buffer, finally resuspending the cells, and repeatedly washing for effectively cleaning the serum on the cell surface to prevent the transfection efficiency from being reduced, but the washing process is gentle and avoids damaging the cells.

(2) Counting cells, taking 10-20 μ l cell suspension, calculating cell concentration, and finally obtaining 1 × 10 cells⁶A tube.

(3) The cells prepared above were mixed with 10. mu.g of the plasmid carrying FOXO1 gene, and the action was gentle to avoid the generation of air bubbles.

(4) Electrotransfection parameters were set according to the cell state, concentration and quality of the plasmid.

(5) Add pre-warmed HuMSCs medium at 37 ℃ to 6 well cell culture plates, 2ml per well.

(6) Add 100. mu.l of the mixture of cells and plasmids to each electric rotor, pump the liquid to notice the bubbles, ensure that each sample has the same resistance value, knock the electric rotor gently to remove the bubbles from the mixture, and then place the electric rotor in the electric rotor holder.

(7) The resistance was measured to ensure that the resistance was between 30-50 Ω and the electrotransfection procedure was started.

(8) And taking out the electric rotating cup, sucking the culture medium in 200-300 mu l of 6-well plate, gently blowing and beating the culture medium and the cell mixed solution uniformly, sucking and transferring all the mixed solution into the 6-well plate, and electrically transfecting the cells in one hole at one time, wherein the culture medium in one hole corresponds to one electric rotating cup.

(9) The above procedure was repeated to perform the electrotransfection of the next well.

Example 3

This example provides a method for introducing the transcription factor FOXO1 into the mesenchymal stem cells by means of lipofection, comprising the following steps:

(1) certain HuMSCs cells are inoculated into a 6-well plate, and transfection is carried out when the cell density is about 70%.

(2) Preparing a transfection mixed solution:

and (3) mixing liquid A: 4. mu.l-8. mu.l Lipofectamine3000+ 250. mu.l Opti-MEM medium;

and (3) mixing liquid B: 3 μ g of plasmid carrying the FOXO1 gene +6 μ l P3000TM +250 μ l of Opti-MEM medium.

And (3) mixing liquid C: the mixed solution A and the mixed solution B are fully mixed, so that repeated blowing is avoided, and the DNA-liposome compound is damaged, so that the transfection efficiency is reduced. Standing at room temperature for 10-15 min to form stable DNA-liposome complex.

(4) Transfection: since starvation of cells can improve transfection efficiency, transfection mixture C was added to cells that had been starved for 1 hour, and then the cells were placed in a 37 ℃ cell incubator for further 8 hours.

(5) Liquid changing: and after 8h of transfection, the HuMSCs culture medium containing FBS is replaced, the HuMSCs culture medium is placed in a cell culture box for continuous incubation for 48h, and the next step of experiment can be carried out after the transfection is finished.

Verification experiment

Carrying out induction culture on the cell strain stably expressing FOXO1 obtained in example 1-3, and detecting the expression levels of cell genes at different time periods by single cell sequencing; detecting the presence or absence of haploid cells using fluorescence hybridization (FISH); detecting the mRNA expression level of the specific marker at different spermatogenesis stages by using RT-qPCR; western Blot is used for detecting the protein expression level of a specific marker at different spermatogenesis stages, and the specific implementation steps are as follows:

single cell sequencing

By utilizing a 10x Genomics chromeum system, Gel Bead with sequence labels, a sample and reagent premixed liquid and oil are loaded to respective sample introduction channels, and a double-cross system formed by a micro-fluid channel network is used for finally forming single-cell micro-reaction system GEMs wrapped by oil drops.

(1) Cell preparation

Performing quality inspection and counting on the single cell suspension, generally requiring the cell survival rate to be more than 80%, washing and re-suspending the qualified cells to prepare cells with proper cell concentration of 700-1200 cells/mu l for 10x genomics chromium^TMAnd operating the system on the computer.

(2) GEM generation and tagging

And (3) according to the number of the expected obtained target cells, constructing GEMs (Gel Bead in Em μ lsion) for single cell separation, and after the GEMs are normally formed, collecting the GEMs and carrying out reverse transcription in a PCR instrument to realize labeling.

(3) Post GEM-RT purification and cDNA amplification

And (3) carrying out oil breaking treatment on the GEMs, purifying and enriching a single-strand cDNA by using magnetic beads, and then carrying out cDNA amplification and quality inspection.

(4) Library construction and quantification

And (3) carrying out second-generation sequencing library construction on the cDNA qualified for quality inspection, and finally carrying out quantitative quality inspection on the library through the experimental processes of fragmentation, connection of a sequencing joint, sample IndexPCR and the like.

(5) Sequencing on machine

Sequencing the constructed library by using an Illumina Hiseq or Novaseq platform and adopting a PE150 sequencing mode, wherein the suggested sequencing quantity reaches 50k reads/cell or above.

FISH

1. Sample processing

(1) Adding 10ml of fresh stationary liquid (3:1 methanol: glacial acetic acid) into the sample, forcibly blowing and uniformly mixing by using a pipette or a pipette, and standing at room temperature for 10 minutes;

(2) centrifuge at 2000 Xg for 5 minutes.

(3) Removing the supernatant, adding 0.5-1 ml of fresh fixing solution into the residual 200 mu l of the mixture, and uniformly blowing and stirring the mixture.

(4) Dripping all the suspension in the centrifugal tube on a clean glass slide (65 ℃), and baking the glass slide for 30 minutes;

2. slide pretreatment

(1) The slides were incubated in 1 × PBS at 37 + -1 deg.C for 5 minutes;

(2) taking out the slide, and then putting the slide into a pepsin solution with the temperature of 37 +/-1 ℃ for digesting for 5 minutes;

(3) the slide is taken out and then is put into 1 XPBS for washing at room temperature for 3 minutes;

(4) taking out the slide, and then placing the slide into 1% paraformaldehyde/PBS for fixing for 10 minutes at room temperature;

(5) the slide is taken out and then is put into 1 XPBS for washing at room temperature for 3 minutes;

(6) the slide is taken out and then is put into 1 XPBS for washing at room temperature for 3 minutes;

(7) taking out the slide, and then putting the slide into 70%, 85% and 100% gradient ethanol for dehydration for 3 minutes respectively;

(8) taking out the slide, and airing the slide at room temperature;

3. simultaneous denaturation of sample and probe, 37 degree hybridization incubation

(1) Taking out the hybridization solution from the kit, shaking and uniformly mixing, and performing instantaneous centrifugation;

(2) adding 10 mul of hybridization solution onto the glass slide, quickly covering the glass slide, and then slightly pressing the glass slide to uniformly distribute the hybridization solution, so as to avoid generating bubbles (if the bubbles are slightly pressed out of the glass slide, the bubbles can cause the failure of hybridization);

(3) mounting a cover glass along the edge of the cover glass by using rubber glue, and completely covering the edge of the cover glass, which is in contact with the glass slide;

(4) placing the slide on a hot table at 78 +/-1 ℃ and performing denaturation for 2min and 30 sec;

(5) quickly placing the slide into a hybridization instrument, and incubating overnight (12-18 hours) at 37 +/-1 ℃;

4. post-hybridization washing and counterstaining

(1) 30 minutes before washing, 0.3NP-40/2 XSSC was placed in a water bath (73 ℃ C.);

(2) taking out the overnight incubated slide in the dark, and carefully removing rubber cement and cover slips;

(3) the slides were quickly placed in a pre-warmed staining jar of 0.3NP-40/2 XSSC and washed at 73 ℃ for 2 minutes;

(4) taking out the slide, putting the slide into a staining jar of 0.1NP-40/2 XSSC, and washing the slide for 30 seconds at normal temperature;

(5) taking out the slide, and dehydrating the slide for 3 minutes respectively by 70 percent of gradient ethanol, 85 percent of gradient ethanol and 100 percent of gradient ethanol at room temperature;

(6) taking out the slide, and airing in the dark;

(7) and (3) dripping 10 mu l of DAPI counterstain to the hybridization area of the glass slide, covering the glass slide, slightly pressing to avoid bubbles, storing in a dark place, and observing.

RT-PCR

Extraction of Total cellular RNA by TRIzol

1.1 sample preparation:

(1) taking out cell culture dish from incubator, with cell fusion rate up to 70-100%, discarding culture medium, washing with DPBS for 1-2 times, and 100mm culture dish²Adding 1ml of TRIzol to lyse the cells, lysing for 10min at room temperature, repeatedly blowing the cells at the bottom of the culture dish by using a 1ml gun head, or repeatedly pumping the cells by using a disposable syringe until the lysate is clear, transparent and non-viscous (the time is about more than 2 min), until the cells are completely dissolved in the TRIzol, and the cell lysate becomes clear and transparent. If lysis is not complete, the amount of TRIzol may be increased appropriately to ensure that all cells must be lysed.

(2) The cell lysate was transferred to a 1.5ml RNase free disposable covered clear polypropylene tube (EP tube), vortexed for 30 seconds, centrifuged at 1000rpm for 5min, the supernatant was transferred to a 1.5ml EP tube, and placed in a-80 ℃ freezer (several hours at room temperature and at least one month at-80 ℃ for storage of the sample), or immediately subjected to the next RNA extraction procedure.

1.2RNA extraction procedure

(1) Chloroform extraction: add 200. mu.l chloroform to 1ml TRIzol, shake the tube vigorously with hand for 20s, incubate at room temperature for 2-3min, centrifuge at 12000rpm at 4 ℃ for 15 min. After centrifugation, the mixture separated into a lower organic phase (bright red lower phase containing proteins, polysaccharides, fatty acids, cell debris and a small amount of DNA), an intermediate phase and an upper colorless aqueous phase, with RNA present in the aqueous phase.

(2) Taking a water phase: transfer the aqueous phase to a new tube. When the water phase is transferred, the gun tip moves downwards along with the downward movement of the liquid level, the phenomenon that layering is disturbed is avoided, the slow suction is carried out, the suction to the intermediate phase and the lower organic phase is avoided, the suction is not too much, and about 400 mu l of water is sucked.

(3) And (3) isopropanol precipitation: adding 600 μ l isopropanol, mixing by turning upside down, standing at room temperature for 10min, centrifuging at 4 deg.C at 12000rpm, and standing for 10 min. Often the RNA was not visible before centrifugation and a clear, whitish precipitate formed on the sides and bottom of the tube after centrifugation.

(4) Ethanol cleaning: the supernatant was discarded, washed once with 1ml of 75% ethanol (at this time, one year at-20 ℃ and one week at 4 ℃), and centrifuged at 7500rpm for 5min at 4 ℃. The RNA pellet was allowed to gently float when 75% ethanol was added, or remained in place, because sometimes the RNA was too small, blowing out RNA debris that could not necessarily have gathered together to form a macroscopic pellet when centrifuged down again, leading to a "blind" procedure later.

(5) Dissolving after drying and precipitating: and (3) absorbing the supernatant as much as possible, drying at room temperature for 5-10min, dissolving with 20-40 mu l of RNase free water, slightly blowing and uniformly mixing, marking, and storing at-80 ℃ or directly carrying out subsequent reverse transcription. During the drying process, the degree of drying of the RNA precipitate is closely noticed, and complete drying is not required, which greatly reduces the solubility of the RNA precipitate. The A260/280 ratio of partially solubilized RNA will be low. The optimal dryness is that the RNA precipitate is slightly moist and gel-shaped.

(6) 2 0.2ml EP tubes are prepared and respectively filled with 1.5 mul of solution dissolved with RNA, and a marking pen, a notebook, a small pipette gun and a small pipette head are arranged for measuring the concentration.

2. Reverse transcription into cDNA

(1) After the components in the GoScript reverse transcription system were taken out and placed on ice to melt, the components were added according to the following table to prepare a reaction solution (the total reaction volume was 20. mu.l, usually, the number of reaction solutions was 1-2 more than that of actual samples to prevent the loss of volume due to liquid wall hanging). After the reaction solution is prepared, the mixture is gently mixed and packaged into a 0.2ml EP tube, and a certain amount of template RNA is added into the primer.

(2) The mixture of template RNA and primers was pre-denatured at 70 ℃ for 5min, and after completion, taken out and placed on ice.

(3) RT-Mix (table below) was prepared on ice, gently mixed and 10. mu.l was added to each sample tube and mixed well.

(4) Setting a reverse transcription program, comprising three steps of annealing, extending and reverse transcriptase inactivation, and specifically comprising the following steps of: 8min at 25 deg.C → 60 mm at 42 deg.C → 15min at 70 deg.C, and finally cooling to 4 deg.C. The cDNA obtained after the reaction is stored in a low-temperature refrigerator at the temperature of-20 ℃ or directly subjected to subsequent operation.

3. Real-time fluorescent quantitative PCR

(1) PCR reaction mixtures (reaction solution preparation can be carried out at room temperature) were prepared according to the following table, and divided into reaction tubes, and then 2. mu.l of template was added:

(2) two-step method qPCR amplification standard procedure.

(3) And (3) data processing and analysis: and (3) after the reaction is finished, confirming an amplification curve and a melting curve of Real Time qPCR, and making a standard curve when PCR quantification is carried out. Taking average Ct value of each sample, usually taking average value of 3-5 multiple wells, taking expression level of Ct of GAPDH as internal control, and adopting 2^-ΔΔCtThe method measures the relative expression level of the gene.

The primers used in the real-time fluorescent PCR are shown in the following table:

WesternBlot

1. sample preparation

(1) Adherent growing cells were removed at different time points, the culture medium was removed and washed gently 2 times with PBS. The PBS was aspirated off and an appropriate amount of pre-cooled lysis buffer was added. The lysate was brought into full contact with the cells by several blows from a gun. After the cells are fully lysed, no cell precipitation is obvious. After full lysis, the sample is collected into a centrifuge tube, centrifuged at 12000rpm at 4 ℃ for 5min, the supernatant is taken for subsequent detection, or the sample is stored at-80 ℃ for later use.

(2) The same amount of protein was taken from each sample, and the same volume of 5 XSDS-PAGE protein loading buffer was added, and the protein sample and the 5 XSDS-PAGE protein loading buffer were mixed. Heating at 100 deg.C or boiling water bath for 5min to fully denature protein, and cooling to room temperature.

2. Electrophoresis

(1) Clean glass plates are prepared and then vertically clamped on a rack for glue pouring. The two glasses are aligned during operation to avoid glue leakage. 10 percent of separation gel is added, and the mixture is immediately shaken up after TEMED is added, so that the gel can be filled. Then a layer of water is added on the glue, and the gelation is quicker after liquid sealing. The glue filling can be started faster, and the speed is slowed down when the glue surface is as high as required. The glue must flow down the glass plate during operation so that no air bubbles are present in the glue. The water sealing is slow when water is added, otherwise the glue is deformed by punching. When there is a line of refraction between the water and the gel, the gel is said to have solidified. Waiting for 3min for the glue to solidify sufficiently, pouring off the water on the upper layer of the glue, and sucking the water with absorbent paper.

(2) 4 percent of concentrated glue is prepared according to the method, and the glue can be filled after being immediately shaken up after TEMED is added. The remaining space was filled with the gel concentrate and a comb was then inserted into the gel concentrate. When the glue is poured, the glue is also made to flow down along the glass plate so as to avoid the generation of air bubbles in the glue. The comb is kept horizontal when inserted. After the concentrated gel is solidified, the two hands respectively hold the two sides of the comb and slightly pull the comb out vertically and upwards.

(3) The concentrated gel was washed with water and placed in an electrophoresis tank.

(4) Sufficient running solution is added and the sample is initially prepared (running solution over at least the small glass plate inside). The sample is sucked up adherently by a microsyringe, 20-40 mug protein is loaded in each sample well, protein Marker is loaded in 1 well, and the sample is sucked out without sucking air bubbles.

(5) Cover electrophoresis tank lid, turn on the power, set up voltage 100V, 2 h.

(6) After electrophoresis, the gel is removed, and the proteins diffuse slowly from the gel and are therefore not stored in the gel, and are then quickly transferred to the next step.

3. Transfer film and seal

(1) And preparing a membrane transferring solution, and soaking the gel in the precooled membrane transferring solution to balance for 5 min.

(2) And (3) shearing a PVDF membrane (which needs to be treated by formaldehyde before use) and filter paper according to the size of the glue, and putting the PVDF membrane and the filter paper into a precooled membrane transferring liquid for balancing for 10 min.

(3) Assembly transfer "sandwich": sponge/3 layers of filter paper/glue/PVDF membrane/3 layers of filter paper/sponge, after each layer is put, the attention is paid that no air bubbles can exist between each layer.

(4) The transfer cell was placed in an ice bath and sandwiched with the membrane near the anode and the gel near the cathode, transfer buffer was added and the electrodes were inserted at 100V for 1h (current approximately 0.3A). Successful transfer of the membrane was marked by the transfer of all bands from the gel to the membrane, and the gel was colorless.

(5) And after the film transfer is finished, turning off the power supply and taking out the PVDF film.

(6) The membrane was immersed in TBST solution, shaken on an orbital shaker at room temperature, and washed 1 time.

(7) The membrane was transferred to blocking solution and blocked by shaking on a decolourisation shaker at room temperature for 1 h.

(8) The membrane was removed and washed 2 times with TBST solution, shaking 5min each time.

4. Antibody incubation and detection

(1) The membrane and primary antibody (dilutions recommended for product application) were incubated at 4 ℃ with occasional gentle shaking overnight.

(2) The membrane was washed three times with TBST solution for 10min each to remove residual primary antibody.

(3) The membrane and secondary antibody (dilutions recommended for product application) were incubated at room temperature for 2 hours with occasional gentle shaking.

(4) The membrane was washed three times with TBST solution for 10min each.

(5) Preparing the developing solution (prepared as before), uniformly coating the developing solution on a film by using a liquid transfer gun, placing the film in a gel image analyzer for photographing, and recording the experimental result.

Results of the experiment

1. Morphological results

As can be seen from fig. 1, the mesenchymal stem cells overexpressing FOXO1 were shortened, broadened, and flattened in morphology, and had a shortened shape, a polygonal shape, and an irregular shape, and had relatively slow growth. The situation that the transcription factor FOXO1 is over-expressed and the proliferation of the mesenchymal stem cells is influenced under the induction environment is shown, and the shape of the mesenchymal stem cells is changed to a certain extent, so that the cells are changed to the triangle-like direction.

2. Single cell sequencing

From fig. 2 to fig. 5, it can be seen that the gene expression levels of germ cell markers (including Primordial Germ Cell (PGC) -specific markers OCT4(POU5F1), SOX17, PRDM1, NANOS3, PRMT5, Spermatogonial Stem Cell (SSCs) -specific markers UCHL1, ID4, DAZL, meiosis markers KIT, SYCP2, SYCP3, DMC1, sperm cell markers ACRV1, SPEM1, TNP1, TXNDC2) at different time points of the mesenchymal stem cells overexpressing FOXO1 were detected and found to be expressed.

FISH results

The probe types are as follows: chromosome 6 centromere (green), chromosome 19 centromere (green), chromosome X centromere (green), and chromosome Y centromere (red).

As can be seen from fig. 6, the detection results show: the signal abnormality is 3 green 1 red blood cells and 2 green 1 red blood cells, namely haploid cells appear.

RT-PCR results

mRNA expression levels of germ cell markers (including Primordial Germ Cell (PGC) -specific markers OCT4 and SOX17, Spermatogonial Stem Cell (SSCs) -specific markers UCHL1 and ID4, meiosis markers KIT and STRA8, sperm cell markers PRM1 and TNP 2.) of the mesenchymal stem cells over-expressing FOXO1 at different time points are detected and are all expressed.

WesternBlot results

As can be seen from fig. 11 to 14, the mesenchymal stem cells (right) overexpressing FOXO1 were cultured for 30 days in comparison with the control group (left), and then the protein expression levels of germ cell markers (including Primitive Germ Cell (PGC) -specific markers OCT4 and SOX17, Spermatogonial Stem Cell (SSCs) -specific markers UCHL1 and ID4, meiosis markers KIT, STRA8, and sperm cell marker TNP2) were measured, and all of them were found to be expressed.

In summary, FOXO1 has an effect of promoting transdifferentiation of mesenchymal stem cells into SSCs, and FOXO1 regulates the differentiation of mesenchymal stem cells into male germ cells, while mesenchymal stem cells express Primitive Germ Cell (PGC) -specific markers OCT4 and SOX17, Spermatogonial Stem Cell (SSCs) -specific markers UCHL1 and ID4, meiosis markers KIT and STRA8, and sperm cell markers PRM1 and TNP2, suggesting that some mesenchymal stem cells undergo meiosis to form male germ cell haploids (sperms).

The embodiments of the present invention have been described in detail, but the embodiments are merely examples, and the present invention is not limited to the embodiments described above. Any equivalent modifications and substitutions to those skilled in the art are also within the scope of the present invention. Accordingly, equivalent changes and modifications made without departing from the spirit and scope of the present invention should be covered by the present invention.

Claims

1. A method for directly transdifferentiating mesenchymal stem cells into sperms by using a transcription factor FOXO1 is characterized by comprising the following specific steps: the transcription factor FOXO1 is introduced into the mesenchymal stem cells by means of transfection, and then the mesenchymal stem cells are induced and cultured into the sperms.

2. The method for directly transdifferentiating mesenchymal stem cells into sperm according to claim 1 using the transcription factor FOXO1, wherein the transcription factor FOXO1 is introduced into the mesenchymal stem cells by means of lentivirus transfection, comprising the following steps:

3. The method for directly transdifferentiating mesenchymal stem cells into sperm according to claim 2, wherein the transfection-accelerating agent in step one is polybrene.

4. The method for directly transdifferentiating mesenchymal stem cells into sperm according to claim 2, wherein the screening medium in step two is a hygromycin B-containing medium.

5. The method for directly transdifferentiating mesenchymal stem cells into sperm according to claim 1 using the transcription factor FOXO1, wherein the transcription factor FOXO1 is introduced into the mesenchymal stem cells by means of electrotransfection, comprising the following steps:

and step two, placing the electric rotating cup in the step one on an electric rotating cup holder, and setting electric transfection parameters for transfection.

6. The method for directly transdifferentiating mesenchymal stem cells into sperm according to claim 1 using the transcription factor FOXO1, wherein the transcription factor FOXO1 is introduced into the mesenchymal stem cells by means of lipofection, comprising the following steps:

step two, adding the mesenchymal stem cells into the transfection mixed solution, and culturing for 7-9h in a cell culture box;

7. The method for directly transdifferentiating mesenchymal stem cells into sperm according to claim 6, wherein the mixture A further comprises Opti-MEM medium in the first step.

8. The method for improving trichoderma liquid fermentation sporulation using light induction as claimed in claim 6, wherein said mixed liquor B in step one further comprises Opti-MEM medium and enhancer.