CN106754661B

CN106754661B - Technical method for differentiating skin stem cells into sperms through ectopic development

Info

Publication number: CN106754661B
Application number: CN201611135047.4A
Authority: CN
Inventors: 沈伟; 孙源超; 程顺峰; 孙晓凤; 李兰
Original assignee: Qingdao Agricultural University
Current assignee: Qingdao Agricultural University
Priority date: 2016-12-11
Filing date: 2016-12-11
Publication date: 2020-04-07
Anticipated expiration: 2036-12-11
Also published as: CN106754661A

Abstract

The invention relates to a method for inducing skin stem cells to differentiate into sperms, which comprises the steps of co-culturing mouse skin stem cells and fibroblasts treated by mitomycin C, inducing the mice skin stem cells to differentiate towards PGC, and reversely injecting PGC cell mixed liquor into testicular seminiferous tubules through receptor mice output tubules; after the mouse testicle grows for one month in the receptor mouse testicle, the receptor mouse testicle is taken out, HE staining analysis finds that spermatogenesis is recovered in the seminiferous tubule, cells with typical sperm morphology appear, and spermatogenic cells in the seminiferous tubule for recovering spermatogenesis express GFP, MVH and the like. The method of the invention has little harm to the organism and no limitation to ethical problems, can effectively induce the skin stem cells to be differentiated into the spermatids, has high differentiation efficiency, and the spermatids obtained by differentiation have typical spermatids and express the specific genes of the spermatids.

Description

Technical method for differentiating skin stem cells into sperms through ectopic development

Technical Field

The invention relates to a method for differentiating skin stem cells into sperms through in vivo development, belonging to the technical field of stem cells and tissue engineering of biomedicine.

Background

In recent years, male infertility patients account for about 10% of the population of the reproductive age of China, and more than 40% of male infertility is caused by abnormal spermatogenesis. Although assisted reproduction techniques bring eosin to many patients, assisted reproduction and drug therapy alone are far from adequate. The method can establish a germ cell generation model in vitro by utilizing the directional differentiation from stem cells to germ cells and finally obtain functional gametes, and has important significance for researching a regulation mechanism of germ cell generation, treating male infertility and the like.

Stem Cells (SC) are a class of cells that have the ability to self-replicate and the potential to differentiate in multiple directions. The stem cells are classified into embryonic stem cells (ES cells) and adult stem cells (stromal cells) according to the developmental stage in which the stem cells are located. Adult stem cells refer to undifferentiated cells present in differentiated tissues, most of which are in a dormant state and maintain their pluripotency, under conditions such that they are capable of self-renewal and differentiation into specific tissue-specific cell types, exhibiting their multipotential differentiation potential. Compared with other adult stem cells, the skin stem cells are widely distributed in the largest organs of human bodies, the material taking is more convenient, and the immune rejection and the ethical problems do not exist in the clinical application, so that the skin-derived stem cells are concerned about differentiating to the germ cells.

However, there are few reports of induced differentiation of skin stem cells to sperm in vitro, and no reports have been reported so far. Other studies of adult stem cell differentiation in vitro into spermatozoa have also mostly come to spermatogonial stem cells. In 1994, Hofmann et al established spermatogonial stem cell lines in vitro and these lines were able to enter meiosis under specific conditions but eventually failed to obtain haploid cells, indicating that this cell line failed to break through the meiotic phase under in vitro culture conditions. In 2002, Feng et al established a cell line in vitro that was able to break through meiosis and produce a roof-like structure upon induction by stem cell factors. But has not been widely used so far because the follow-up is not repeated by other laboratories.

Disclosure of Invention

In order to solve the defects and raise the advantages in the prior art, the invention aims to provide a method for differentiating mouse skin stem cells into sperms through in vivo development by taking the skin stem cells as primitive cells for the first time, wherein the mouse skin stem cells have little harm to organisms and are not limited by ethical problems.

In order to achieve the purpose, the invention adopts the following technical scheme: a method for differentiating a skin stem cell into a sperm through in vivo development comprises the two steps of inducing and differentiating the skin stem cell into a primordial germ cell and implanting the primordial germ cell into a receptor mouse testis to obtain the sperm cell, wherein:

1) the induced differentiation of skin stem cells into primordial germ cells is specifically as follows:

① preparing skin stem cells, namely extracting skin stem cells of a mouse cultured with 1-2 generations of green fluorescence transfer genes and wild mouse fibroblasts respectively, treating the wild mouse fibroblasts with mitomycin C, and mixing the skin stem cells and the treated fibroblasts according to the mass ratio of 1:1 for later use;

② DMEM high-sugar culture medium is selected as the primary germ cell induction culture medium, fetal bovine serum with mass fraction of 8% -12% of total mass of DMEM high-sugar, 40 mIU/ml-60 mIU/ml follicle stimulating hormone, 80 IU/ml-120 IU/ml leukemia inhibiting factor, 15 ng/ml-25 ng/ml bone morphogenetic factor, 0.20 mmol/L-0.25 mmol/L sodium pyruvate, 0.08 mmol/L-0.12 mmol/L nonessential amino acid, 1.5 mmol/L-2.5 mmol/L L-glutamine, 0.08 mmol/L-0.12 mmol/L β -mercaptoethanol, 8 ng/ml-12 ng/ml epidermal growth factor, 15 ng/ml-25 ng/ml basic fibroblast growth factor, 30 ng/ml-50 ng/ml dry cell growth factor, culture temperature 35 ℃ -38 ℃ and humidity not lower than 80% of CO in environment are added into the culture medium₂The concentration is 4% -6%;

③ implanting the mixed cells obtained in step ① into the primordial germ cell induction culture medium prepared in step ②, and culturing the culture medium in an incubator for 8-10 days in a conventional manner to obtain induced and differentiated primordial germ cells;

2) the method for implanting primordial germ cells into receptor mouse testis to obtain sperm cells specifically comprises the following steps:

① preparing a recipient mouse;

② diluting the primordial germ cells obtained in step ③ of 1) to 0.8X 10⁶Per liter of 1.2 x 10⁶And (3) per L, injecting the cells into a receptor testicle seminiferous tubule by using a conventional testicle injection technology, and culturing until cells with typical sperm morphology are detected by a conventional method, wherein the cells with the typical sperm morphology are the required sperm cells.

The method for differentiating the skin stem cells into sperms through in vivo development comprises the step 1) of inducing and differentiating the skin stem cells into primordial germ cells and implanting the primordial germ cells into receptor mouse testes to obtain sperms, and the step of culturing the primordial germ cells specially comprises the step of placing the primordial germ cells obtained in the step ③ in the step 1) into a specially-oriented culture medium for culturing, wherein the specially-oriented culture medium is prepared by taking a DMEM high-sugar medium as a base, adding fetal calf serum accounting for 8% -12% of the total mass of the DMEM high-sugar, bone morphogenetic factors accounting for 15 ng/ml-25 ng/ml, bone morphogenetic factors accounting for 0.20 mmol/L-0.25 mmol/L sodium pyruvate, nonessential amino acids accounting for 0.08 mmol/L-0.12 mmol/L, amino acids accounting for 1.5 mmol/L-2.5 mmol/L L-glutamine, basic fibroblast growth factors accounting for 0.08 mmol/L-0.12 mmol/L β -mercaptoethanol, epidermal growth factors accounting for 15 ng/ml-25 ng/ml, epidermal growth factors accounting for 30ng/ml, basic fibroblast growth factors for 50-35-ml, and culturing for 50-35-day₂The concentration is 4-6%.

The method for differentiating the skin stem cell into the sperm through in vivo development comprises the following steps: the period of incubation of primordial germ cells in the testes of the recipient mouse is 25-40 days.

Compared with the prior art, the invention has the following advantages: the mouse skin stem cells and fibroblasts are used as protocells for directional induced differentiation, on one hand, the damage to organisms is small, on the other hand, the limitation of ethical problems is avoided, and the method has wider application prospect and great social and economic significance; the adopted culture medium has more reasonable component configuration, more complete nutrition and more special direction, and has high efficiency, high reproduction rate, high purity and good quality for directionally culturing the spermatids by the applicable skin stem cells; the initial induced differentiation and the final in vivo culture are clearly divided, so that on one hand, the specificity of the method is stronger, on the other hand, larger space and application value are provided for the optimization of the steps of practical application, the culture difficulty is greatly reduced, and the reproducibility is improved; the invention establishes a method for differentiating mouse skin stem cells into sperms through in vivo development, takes the mouse skin stem cells as a material, firstly induces and differentiates the mouse skin stem cells into primordial germ cells in vitro, differentiates the primordial germ cells into sperms with typical sperm morphology by utilizing the in vivo environment through a testis injection technology, and provides a technical basis for the differentiation of the skin stem cells into gametes and the research and application.

Drawings

FIG. 1 is a picture of mouse skin stem cell culture and passage.

FIG. 2 is a picture of primordial germ cells of mouse skin stem cell differentiation.

Fig. 3 is a picture of testis from normal mice and busulfan treated mice.

FIG. 4 is a picture of the primordial germ cells differentiated from mouse skin stem cells injected into recipient mouse testis.

FIG. 5 is a photograph showing HE staining of recipient mouse testis tissue section.

FIG. 6 is an immunofluorescence histochemical staining image of a recipient mouse testis section.

Detailed Description

The process according to the invention is further illustrated below by means of specific examples and figures.

Example 1

A method for differentiating a skin stem cell into a sperm through in vivo development comprises the two steps of inducing and differentiating the skin stem cell into a primordial germ cell and implanting the primordial germ cell into a receptor mouse testis to obtain the sperm cell, wherein:

① preparing skin stem cell, extracting skin stem cell and wild mouse fibroblast of mouse cultured with 1 generation of green fluorescence transfer gene, treating wild mouse fibroblast with mitomycin C, and mixing skin stem cell and treated fibroblast at a mass ratio of 1: 1;

② DMEM high-sugar culture medium is selected as the primary germ cell induction culture medium, fetal calf serum with 8% mass fraction calculated by the total mass of DMEM high sugar in the culture medium, follicle stimulating hormone 40 mIU/ml calculated by the total volume of DMEM high sugar in the culture medium, leukemia inhibiting factor 80 IU/ml, bone morphogenetic factor 15ng/ml, sodium pyruvate 0.20mmol/L, nonessential amino acid 0.08 mmol/L, glutamine 1.5mmol/L L, fibroblast growth factor 0.08 mmol/L β -mercaptoethanol, epidermal growth factor 8ng/ml, alkaline fibroblast growth factor 15ng/ml, fibroblast growth factor 30ng/ml, culture temperature 35 ℃, humidity 80%, and CO2 concentration 4% in the environment are added in the culture medium;

① preparing a recipient mouse;

② diluting the primordial germ cells obtained in step ③ of 1) to 0.8X 10⁶And (4) per L, injecting the cells into a receptor testis seminiferous tubule by using a conventional testis injection technology, and detecting the cells with the typical sperm morphology by a conventional method after 40 days, wherein the cells with the typical sperm morphology are the required sperm cells.

Example 2

A method for differentiating a skin stem cell into a sperm through in vivo development comprises the three steps of inducing and differentiating the skin stem cell into a primordial germ cell, culturing the primordial germ cell in a special direction, and implanting the primordial germ cell into a receptor mouse testis to obtain the sperm cell, wherein:

① preparing skin stem cells, namely extracting skin stem cells cultured with 2-generation green fluorescence transfer gene mice and wild mouse fibroblasts respectively, treating the wild mouse fibroblasts with mitomycin C, and mixing the skin stem cells and the treated fibroblasts according to the mass ratio of 1:1 for later use;

② DMEM high-sugar culture medium is selected as the primary germ cell induction culture medium, and fetal calf serum with the mass fraction of 12% calculated by the total mass of DMEM high sugar in the culture medium, 60 mIU/ml follicle-stimulating hormone, 120IU/ml leukemia inhibitory factor, 25ng/ml bone morphogenetic factor, 0.25 mmol/L sodium pyruvate, 0.12mmol/L nonessential amino acid, 2.5mmol/L L-glutamine, 0.12mmol/L β -mercaptoethanol, 12ng/ml epidermal growth factor, 25ng/ml alkaline fibroblast growth factor, 50ng/ml dry cell growth factor are added into the culture medium, the culture temperature is 38 ℃, the humidity is 85%, and the concentration of CO2 in the environment is 6%;

2) the specific culture of primordial germ cells comprises the following steps:

placing the primordial germ cells obtained in the step ③ in the step 1) into a special culture medium for culturing, wherein the special culture medium is specifically characterized in that a DMEM high-sugar medium is taken as a base, fetal calf serum accounting for 8% of the total mass of the DMEM high-sugar in the medium, bone morphogenetic factor accounting for 15ng/ml of the total volume of the DMEM high-sugar in the medium, sodium pyruvate accounting for 0.20mmol/L, nonessential amino acid accounting for 0.08 mmol/L, 1.5mmol/L L-glutamine, 0.08 mmol/L β -mercaptoethanol, epidermal growth factor accounting for 15ng/ml, alkaline fibroblast growth factor accounting for 30ng/ml, dry cell growth factor accounting for 3 days, the temperature is 35 ℃, the humidity is 80%, and CO in the environment₂The concentration is 4%; after a certain time of special culture, more purified, more numerous and better-shaped primordial germ cells can be obtained.

3) The method for implanting primordial germ cells into receptor mouse testis to obtain sperm cells specifically comprises the following steps:

① preparing a recipient mouse;

② diluting the primordial germ cells obtained in 2) to 1.2X 10⁶Per liter, then injected using conventional testicular injection techniquesAnd (3) detecting the cells with the typical sperm morphology in the testes, namely the cells with the typical sperm morphology are the required sperm cells by a conventional method after 25 days.

Example 3

The same as example 2, except that:

2) the specific culture of primordial germ cells comprises the following steps:

placing the primordial germ cells obtained in the step ③ in the step 1) into a special culture medium for culturing, wherein the special culture medium is prepared by taking a DMEM high-sugar medium as a base, adding 12% of fetal calf serum by mass percent of the total mass of the DMEM high-sugar in the medium, 25ng/ml of bone morphogenetic factor, 0.25 mmol/L of sodium pyruvate, 0.12mmol/L of nonessential amino acid, 2.5mmol/L L-glutamine, 0.12mmol/L of β -mercaptoethanol, 25ng/ml of epidermal growth factor, 50ng/ml of alkaline fibroblast growth factor and 50ng/ml of dry cell growth factor, and culturing for 5 days at the temperature of 38 ℃ and the humidity of 85% in an environment with CO₂The concentration is 6 percent; after a certain time of special culture, more purified, more numerous and better-shaped primordial germ cells can be obtained.

Example 4

This example describes the steps of the method of the present invention in more detail, in conjunction with the pictures, specific operation procedures, instruments used, sources of specific drugs, etc., for explaining the method of differentiating the skin stem cells into sperm through in vivo development, as follows:

1. isolation of mouse skin Stem cells

The newborn green fluorescent transgenic CD1 male mice on the day are taken, and after the neck is cut off, the mice are wiped clean by 75% alcohol and then washed by normal saline once. The skin was cut along the outer side of the mouse back with scissors, the back skin was peeled off with forceps and placed in DMEM/F12, and the skin was stripped of fat and blood with curved forceps. The skin was washed 3 times in 2-3 mL of Phosphate Buffered Saline (PBS) containing 1% streptomycin (PS), and then transferred to a 6cm dish in groups of 5-7 mice each. Cut the skin of the mouse into pieces with scissors<1mm 2 chips, then turnThe cells were transferred to a 15 mL centrifuge tube, added PBS to 10 mL, centrifuged at 1200 rpm for 3min, and washed 3 times. The supernatant was discarded, 1 mL of 0.25% pancreatin was added, and the mixture was left at 37 ℃ under saturated humidity and 5% CO₂Digesting in incubator for 20-30 min. The centrifuge tube was removed, 1 mL of 0.1% DNase I was added to each tube and incubated at room temperature for 1 min. 9 mLPBS was added, the cells were resuspended and centrifuged at 1200 rpm for 3 min. The supernatant was discarded, 10 mL of PBS was added, the cells were resuspended, and centrifuged at 1200 rpm for 3 min. The supernatant was discarded, 10 mL of DMEM/F12 was added, the cells were resuspended, centrifuged at 1200 rpm for 3min, and the washing was repeated 3 times. The supernatant was discarded, 1 mL of DMEM/F12 was added, and the tissue was tapped using a pipette tip with the tip until the tissue suspension was free to aspirate into the tip. 10 mL of DMEM/F12 was added, the cells were resuspended, and the tissue was filtered using a 40 um disposable cell sieve, the cells were collected in a 50mL centrifuge tube and centrifuged at 1500rpm for 5 min. The supernatant was discarded, 10 mL of stem cell medium was added to resuspend the cells and transferred to a 10 cm suspension culture dish. At this time, the cells were designated as P0 generation. The mouse skin stem cell culture medium contains DMEM/F12; 2% B27; 20ng/ml epidermal growth factor; 40ng/ml basic fibroblast growth factor; 1% streptomycin qing; the day of culture was zero passage, and the passage was once every 4 days. In FIG. 1, (1) is a freshly cultured skin stem cell suspension, similar cell aggregates appear on the next day of culture (2) in FIG. 1), and the skin stem cell clone spheres are clear in edge and compact on the fourth day (3 in FIG. 1. the number of hybrid cells decreases after passage, and the number of skin stem cell clone spheres increases (4 in FIG. 1).

2. Induction of mouse Primordial Germ Cells (PGCs)

Collecting 1-2 generation skin stem cell clone ball, digesting with 0.25% Trypsin, blowing to single cell with gun in room temperature digestion process, stopping digestion with 10% fetal calf serum, collecting cell, centrifuging at 1500rpm for 5 min; discarding supernatant, adding small amount of primordial germ cell induction culture medium to suspend cells, and diluting cells to cell concentration of 5 × 10⁴Per ml primordial germ cell induction culture medium;

collecting 1-3 generation fibroblast, pouring off culture medium, adding 4ml fibroblast culture medium containing 10 μ g mitomycin C, placing in incubator for 1.5-2 hr, taking out, washing with phosphate buffer solutionDigesting adherent fibroblasts with 0.25% Trypsin for 5 times, washing, adding primordial germ cell induction culture medium, and diluting to 5 × 10⁴Mixing the two cell mixed solutions at a ratio of 1:1, adding into 6cm primordial germ cell induction culture medium, and placing at 37 deg.C with saturated humidity and 5% CO₂Culturing, wherein the primordial germ cell induction medium comprises DMEM high sugar, 10% fetal calf serum, 50 mIU/ml follitropin, 100 IU/ml leukemia inhibitory factor, 20ng/ml bone morphogenetic factor (BMP-4), 0.23 mmol/L sodium pyruvate, 0.1mmol/L nonessential amino acids, 2mmol/L L-glutamine, 0.1mmol/L β -mercaptoethanol, 10ng/ml epidermal growth factor, 20ng/ml basic fibroblast growth factor, 40ng/ml stem cell growth factor, wherein the cell suspension begins to be in suspension (1) in FIG. 2), substantially adheres on the next day (2) in FIG. 2, and cells with large refractivity appear on 8-10 days in culture, (3) in FIG. 2), wherein the primordial germ cell induction medium is replaced by a composition comprising DMEM high sugar, 10% fetal calf serum, 0.23 mmol/L sodium pyruvate, 0.1mmol/L nonessential amino acids, 2mmol/L sodium pyruvate, 0.83 ng/L sodium pyruvate, 0.1 mmol/ml stem cell growth factor, 40ng/ml stem cell growth factor (20 ml), and the number of the growth factor increases with the addition of BMP-40 ml growth factor (20 ml).

3. Receptor mouse testis injection

Primordial germ cells were collected and washed, stained with 0.1% trypan blue, and placed on ice. Selecting a good receptor mouse, treating the receptor mouse with busulfan, and injecting sodium pentobarbital into the abdominal cavity to perform anesthesia, wherein the spermatogenic epithelium of the receptor mouse is destroyed (2) in figure 3) compared with that of a normal mouse (1 in figure 3). Dosage: 240 mg/kg. After the mice were comatose, they were supine and the limbs were secured to a cardboard with tape. The ventral skin of the mice was moistened with alcohol and the ventral hairs of the mice were cut off. The skin, peritoneum and abdominal muscles are sequentially cut from bottom to top in the abdomen of the receptor mouse, and the incision is about 0.5cm away from the genitals and about 1 cm long. Pull the testis out of the abdomen with forceps: the fat pad connected to the testis was found beside the bladder and gently pulled out of the abdominal cavity, and the testis and epididymis of the mouse were pulled out of the abdominal cavity. Finding the output tubule of the receptor mouse under the stereoscope, and adjusting the angle of the output tubule to ensure that the direction of the output tubule of the receptor mouse is approximately parallel to the direction of the injection needle. The two hands operate, clamp the output small tube, put one section into the head of the glass needle, and move the output tube to the direction of the glass needle. After the needle is inserted in place, the piston is pushed slowly to inject the original germ cell fluid into the receptor mouse's seminal tubule. The testis, epididymis and their fat are slowly delivered into the abdominal cavity and placed in situ, and the abdominal muscle and skin are sutured in turn. After suturing, the mice were sterilized with 75% alcohol and iodine tincture in order, and placed in warm areas, and returned to the laminar flow frame for sterile culture after they were awakened. One month after testicular injection, recipient mouse testis was removed for further analysis. FIG. 4 shows the primordial germ cells differentiated from mouse skin stem cells injected into recipient mouse testicular seminal tubules.

4. Receptor mouse testis HE section staining observation tissue structure

Taking out testes of a receptor mouse, fixing the testes overnight by 4% paraformaldehyde, washing, dehydrating, clearing, embedding paraffin, slicing tissues, removing paraffin on the slices by xylene twice in each step for 10min, and descending to water by absolute alcohol and alcohol at each stage. Hematoxylin solution staining was for about 5 minutes. Putting the slices into a tap water dye vat, and replacing the dye vat until the tap water does not change color. Adding 70% ethanol solution of 0.5% hydrochloric acid (1.5% hydrochloric acid ethanol for short) and color separating. During color separation, the color is separated while microscopic examination is carried out until cell nuclei are clearly stained, and the cell nuclei are dipped in hydrochloric acid alcohol for three times in general, wherein each time is not more than 1 s. Color separation was followed by basification with tap water until the nuclei became blue. This step may also be referred to as "bluing". Washing with distilled water to remove alkaline components. If too much alkaline substance remains in the section, the subsequent eosin staining is not favorable. Adding 50%, 70% and 80% alcohol in sequence. Put into 1% eosin alcohol for 2 minutes. Eosin was fractionated with 95% alcohol until the cytoplasm and connective tissue appeared pink. It is usually 1 min. Adding into 95% alcohol, and dehydrating with anhydrous alcohol. The time per step was 20 s. Adding xylene for transparency (about 15 min). Taking out the section, wiping off excessive xylene around the tissue, dripping gum, and sealing with a cover glass. By HE staining, it was found that most of the seminiferous tubules in the recipient mouse testis were still deficient in spermatogenesis, but some of the seminiferous tubules had restored spermatogenesis (fig. 5).

5. Receptor mouse testis immunofluorescence histochemical detection of specific gene expression

Taking out the testes of a receptor mouse, fixing the testes overnight by 4% paraformaldehyde, washing, dehydrating, clearing, embedding paraffin, slicing tissues, and then putting the cut paraffin slices, namely white slices, into a 60 ℃ oven to be dried for 2 hours. Paraffin on sections was stripped off twice in xylene for 15min each time. Dewaxing, descending to water through absolute alcohol and alcohol at each level, putting the rehydrated slices into citric acid antigen repairing liquid, and repairing for 15min at 96 ℃. After the repair liquid is cooled to room temperature, the sheet is taken out and placed in a horizontal wet box, a pencil is used for marking the tissue section area, and then the sheet is placed in TBS for washing for 5min and then is transferred to TBST for washing for 5 min. BDT (3% BSA and 10% goat serum in TBS) was added to block approximately 100. mu.L per tablet. The sealing film is then applied to the section area. Standing at room temperature for 30 min. And removing the sealing film, and pouring the residual sealing liquid on the sheet onto the absorbent paper. Add appropriate amount of primary antibody to the slice position, typically 20. mu.L. Sealing the primary antibody with a sealing film, taking care that no air bubbles are present, especially at the tissue section site. The slide glass is put into a wet box, sealed by a sealing bag and incubated for 1 h at 37 ℃. After the primary antibody incubation was complete, the discs were removed and the discs were soaked in TBST. Three times for 10min each. TBST from the slide is poured onto absorbent paper and a fluorescently labeled secondary antibody is added, typically in an amount of 20. mu.L. Sealing the secondary antibody with sealing film, placing into a wet box, sealing with sealing bag, and incubating at 37 deg.C for 15 min. After the incubation is finished, the sheet is taken out, the sealing film is removed, and TBST is used for washing for three times, namely 10min, 20min and 20 min. Pouring the redundant TBST on the slice onto absorbent paper, dripping a proper amount of Hoechst 33342 (generally 10 mu L), adding a sealing film at the slice position, incubating at normal temperature for 3min, and controlling the staining time, wherein the staining of the slice is obviously faster than that of the cell. The sealing film was removed and washed with PBS 3 times for 5min each. Excess PBS was poured onto absorbent paper. And adding Vectashield sealing sheets, wherein the adding amount depends on the area of the tissues on the sheets, and generally 5-10 mu L of the sealing sheets is added. Care was taken not to drip directly onto the tissue, but rather should drip onto the tissue slice edge and cover the slice with a coverslip. Placing the prepared slices in a dry box, covering a sealing bag to prevent the slices from being condensed with water, storing at 4 ℃, and observing by microscopic examination. Note that it cannot be placed in a wet box. As a result of staining, in seminiferous tubules in the recipient testis, which restore spermatogenesis, spermatogenic epithelial cells express both GFP (1-2 in FIG. 6) and MVH (1-3 in FIG. 6), indicating that the cells restoring spermatogenesis are derived from mouse skin stem cell differentiated primordial germ cells.

The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for differentiating a skin stem cell into a sperm through in vivo development comprises two steps of inducing and differentiating the skin stem cell into a primordial germ cell and implanting the primordial germ cell into a receptor mouse testis to obtain the sperm cell, and is characterized in that:

② DMEM high-sugar culture medium is used as primordial germ cell induction culture medium, and DMEM high-sugar is added into the culture medium8-12% of fetal bovine serum by mass percentage based on the total mass, 40-60 mIU/ml of follicle-stimulating hormone, 80-120 IU/ml of leukemia inhibitory factor, 15-25 ng/ml of bone morphogenetic factor, 0.20-0.25 mmol/L of sodium pyruvate, 0.08-0.12 mmol/L of nonessential amino acid, 1.5-2.5 mmol/L of L L-glutamine, 0.08-0.12 mmol/L of β -mercaptoethanol, 8-12 ng/ml of epidermal growth factor, 15-25 ng/ml of basic fibroblast growth factor, 30-50 ng/ml of dry cell growth factor, 35-38 ℃ of culture temperature, 80% of humidity and CO in environment₂The concentration is 4% -6%;

① preparing a recipient mouse;

2. The method for differentiating the skin stem cells into the sperms through the in vivo development according to claim 1, which is characterized in that a special primordial germ cell culture step is added between the step 1) of inducing the skin stem cells into the primordial germ cells and the step of implanting the primordial germ cells into recipient mouse testis to obtain the sperms, specifically, the step ③ of the step 1) of obtaining the primordial germ cells is placed into a special culture medium for culture, and the special culture medium is specifically characterized in that a DMEM high-sugar medium is used as a base, and fetal bovine serum with mass fraction of 8% -12% of the total mass of DMEM high-sugar, bone morphogenetic factors with the total volume of DMEM high-sugar, and 0.20mmol of the fetal bovine serum are added into the special culture medium for culture, wherein the special culture medium is formed by taking the DMESodium pyruvate of 0.25 mmol/L, nonessential amino acid of 0.08 mmol/L to 0.12mmol/L, 1.5mmol/L to 2.5mmol/L L L-glutamine, β -mercaptoethanol of 0.08 mmol/L to 0.12mmol/L, epidermal growth factor of 15ng/ml to 25ng/ml, basic fibroblast growth factor of 30ng/ml to 50ng/ml, dry cell growth factor of 30ng/ml to 50ng/ml, culturing for 3 to 5 days at 35 ℃ to 38 ℃ and with the humidity not lower than 80 percent, and CO in the environment₂The concentration is 4-6%.

3. The method of claim 1, wherein the method comprises the step of differentiating the skin stem cell into the sperm through in vivo development: the period of incubation of primordial germ cells in the testes of the recipient mouse is 25-40 days.