Spermatogonial stem cell line and preparation method thereof

Abstract

The invention provides a method for preparing a spermatogonial stem cell line from a mammal individual. The invention also provides the spermatogonial stem cell line prepared by the method.

Description

Technical Field

[0001] The present invention relates to the field of stem cells. Specifically, the present invention provides a spermatogonial stem cell line and a method for preparing the same. Background Art

[0002] Spermatogonial Stem Cells (SSCs) are undifferentiated germ cells located in the basal layer of the seminiferous tubules of the testis, and have the dual abilities of self-renewal and directional differentiation into sperm. As the core cell population in male germline development, SSCs maintain the homeostasis of the stem cell pool through asymmetric division, and at the same time generate differentiated spermatogonial progenitor cells (SPCs), and finally form mature gametes through meiosis. The marker molecules of SSCs include PLZF, GFRα1, RET, NANOS2, etc.

[0003] Establishing a stable spermatogonial stem cell line is of great significance for biological research work, including being used to study the mechanism of male infertility and the development of reproductive technologies. The in vitro culture of SSCs provides a model for analyzing the molecular mechanism of spermatogenic disorders (such as non-obstructive azoospermia). The stable SSC line can be used for gene editing (such as CRISPR / Cas9-mediated genetic modification) and autologous transplantation, providing strategies for hereditary infertility or fertility preservation after radiotherapy and chemotherapy. In addition, the spermatogonial stem cell line can also be used as a toxicity evaluation platform to evaluate the potential harm of environmental toxins or drugs to the reproductive system by monitoring the proliferation and differentiation ability of SSCs.

[0004] Existing studies have established long-term spermatogonial stem cell lines of economic fish (such as Opsariichthys bidens), solving the problem of short in vitro survival time of fish stem cells. Compared with fish, the preparation and culture of mammalian spermatogonial stem cells require more complex microenvironment support.

[0005] There is still a need in this field for a more extensive study on the stemness mechanism of mammalian spermatogonial stem cells, and to develop methods and culture media for stably growing and maintaining the stemness of spermatogonial stem cell lines in vitro. Summary of the Invention

[0006] The present application provides a new method for culturing spermatogonial stem cell lines. Through the method of the present invention, a spermatogonial stem cell line that can grow stably and maintain its stemness for a long time can be generated.

[0007] Specifically, the present invention provides a method for preparing a mammalian spermatogonial stem cell line, which comprises the following steps:

[0008] Step 1: Purify and culture spermatogonial stem cells from the testis of a mammal;

[0009] Step 2: Subculture the spermatogonial stem cells obtained in Step 1 for 1 to 3 passages;

[0010] Step 3: Subculture the spermatogonial stem cells obtained in Step 2 in a feeder layer (such as an embryonic fibroblast feeder layer). After multiple passages of subculture, a spermatogonial stem cell line is obtained.

[0011] In one aspect of the present invention, Step 1 in the method includes the following steps:

[0012] (1) Digest the testicular tissue with collagenase;

[0013] (2) Digest with trypsin;

[0014] (3) Purification: Remove somatic cells to obtain spermatogonial stem cells;

[0015] (4) Culture the obtained spermatogonial stem cells in a spermatogonial stem cell medium.

[0016] In one aspect of the present invention, Step 1 in the foregoing method includes the following steps:

[0017] (1) Testis isolation: Isolate and obtain testes from a mammal such as a mouse;

[0018] (2) Collagenase digestion, for example, digest in 1 mg / mL collagenase for about 10 - 20 minutes;

[0019] (3) Trypsin digestion, for example, after collagenase digestion, digest with 0.05% trypsin for about 2 - 8 minutes, and then terminate the digestion;

[0020] (4) Purification: Remove somatic cells by differential adhesion to obtain spermatogonial stem cells, and culture them in a spermatogonial stem cell medium.

[0021] In the present invention, enriched or purified spermatogonial stem cells derived from the testis can be obtained through the said Step 1.

[0022] In one aspect of the present invention, the subculture in Step 2 is carried out on the spermatogonial stem cells purified in Step 1 after culturing for 15 - 30 days.

[0023] In one aspect of the present invention, the subculture in Step 2 is carried out on the spermatogonial stem cells purified in Step 1 after culturing until the clone mass density is greater than 60%, preferably greater than 80%, more preferably greater than 90%.

[0024] In one aspect of the present invention, the subculture in Step 2 is carried out for 1 - 3 passages, preferably 3 passages.

[0025] In one aspect of the present invention, the passage culture in step 3 of the foregoing method is carried out for more than 20 passages, preferably for more than 30 passages.

[0026] In one aspect of the present invention, the density of the feeder layer (such as an embryonic fibroblast feeder layer) in the foregoing method is greater than 80%, preferably greater than 90%, and more preferably greater than 95%.

[0027] In the present invention, in the foregoing method, the spermatogonial stem cell medium is a medium (or culture solution) suitable for culturing stem cells, which includes maintaining the proliferation ability, developmental potential and stemness of stem cells, etc. The stem cell medium that can be used in the present invention can be provided and obtained in a commercially available form. The medium that can be used in the present invention can be provided and obtained in a commercially available form.

[0028] In one aspect of the present invention, the spermatogonial stem cell medium contains various basal media or nutrient media suitable for culturing cells, especially for the growth of stem cells. The basal medium or nutrient medium is a cell culture medium containing nutrients that promote cell proliferation, generally containing isotonic saline, buffer solution, protein source (in the form of one or more added proteins or amino acids) and other externally added nutrients and growth factors. The basal medium or nutrient medium that can be used in the present invention can be provided and obtained in a commercially available form, for example, including DMEM (Dulbecco's Modified Eagle Medium), KO DMEM (Knockout DMEM), F12 / 50% DMEM, StemPro-34 SFM, StemPro-34 supplement, etc.

[0029] The substances that can promote cell growth in the spermatogonial stem cell medium of the present invention include transferrin, sodium pyruvate, progesterone, β-estradiol, β-mercaptoethanol, etc. The growth factors that can be used in the spermatogonial stem cell medium of the present invention include cytokines such as epidermal growth factor EGF, glial cell line-derived neurotrophic factor GDNF, basic fibroblast growth factor bFGF, leukemia inhibitory factor LIF, etc. It should be noted that the spermatogonial stem cell medium of the present invention does not include insulin or insulin-like growth factor-1. The inventors unexpectedly found that in the method for establishing a stable spermatogonial stem cell line in the present invention, adding insulin to the medium for culturing spermatogonial stem cells will cause damage to the growth of spermatogonial stem cells (the typical phenomenon is that the stem cells shrink) and even cause the death of spermatogonial stem cells.

[0030] In one aspect of the present invention, the stem cell culture solution in the foregoing method contains fetal bovine serum (FBS). In one aspect of the present invention, the stem cell culture solution in the foregoing method contains vitamins.

[0031] In one aspect of the present invention, the stem cell culture medium in the aforementioned method further contains non-essential amino acids, such as one or more of glycine, alanine, asparagine, aspartic acid, glutamine / glutamic acid tyrosine, proline, and serine.

[0032] In one aspect of the present invention, the stem cell culture medium in the aforementioned method further contains antibiotics.

[0033] In one aspect of the present invention, the spermatogonial stem cell medium comprises: StemPro34 SFM base + 1xStemPro34 nutrient supplement + 5mg / mL bovine serum albumin BSA + 6mg / mL glucose + 2mM GlutaMAX + 1x non-essential amino acids NEAA + 1x penicillin-streptomycin double antibody + 1x MEM vitamins + 10μg / mL biotin d-Biotin + 30μg / mL sodium pyruvate + 0.06% lactic acid + 100μM ascorbic acid + 30nM sodium selenite + 60μM putrescine + 100μg / mL transferrin + 60ng / mL progesterone + 30ng / mL β-estradiol + 10μM β-mercaptoethanol + 1% FBS + 20ng / mL EGF + 20ng / mL GDNF + 10ng / mL bFGF + 1x10 3 U / mL LIF.

[0034] In one aspect of the present invention, the method for preparing a mammalian spermatogonial stem cell line provided includes the following steps:

[0035] Step 1: Isolate, purify and culture spermatogonial stem cells from the testis;

[0036] (1) Testis treatment: Obtain testes from mammals such as mice, remove blood cells with a buffer, remove the tunica albuginea on the surface of the testes, and divide the testis tissue into small pieces;

[0037] (2) Collagenase digestion: Transfer the small pieces of testis tissue to 1mg / mL collagenase and digest at about 37°C for about 15 minutes;

[0038] (3) Trypsin digestion: After collagenase digestion, digest with 0.05% trypsin at about 37°C for about 5 minutes, and then add 10% FBS or 5 - 8mL of MEF medium containing 10% FBS to terminate the digestion;

[0039] (4) Purification: After filtering with a 40μm cell sieve, collect the filtrate and centrifuge, discard the supernatant, then resuspend the cell pellet with SSCs medium, then inoculate the cell suspension into the wells of a culture plate, perform differential adhesion overnight, the next day, gently pipette the bottom of the culture plate, collect the supernatant for culture, and change the medium by half every 3 days;

[0040] Step 2: Subculture the spermatogonial stem cells obtained in Step 1 for 1 - 3 passages, preferably for 3 passages.

[0041] Step 3: Subculture the spermatogonial stem cells obtained in Step 2 in a feeder layer of embryonic fibroblasts.

[0042] The present invention also provides a mammalian spermatogonial stem cell line. In one embodiment, the mammalian spermatogonial stem cell line is prepared by the aforementioned method. Brief Description of the Drawings

[0043] Figure 1 Showing exemplary subculture of spermatogonial stem cells to obtain a spermatogonial stem cell line by the method of the present invention: Under an optical microscope, the spermatogonial stem cell clone clusters are in the shape of grape clusters, with a smooth outer periphery, large cell bodies, about 10 μm in diameter, and relatively large nuclei, which are round or oval. In the early stage of cell culture (P1 generation), the SSCs proliferate slowly. By the 10 - 12th day of culture, only a small number of clone clusters appear. With the increase in the number of passages (P33), the proliferation rate of SSCs accelerates.

[0044] Figure 2 Showing the identification and analysis of the spermatogonial stem cell line prepared by the method provided by the present invention using immunofluorescence staining.

[0045] Figure 3 Showing the identification and analysis of the spermatogonial stem cell line prepared by the method provided by the present invention using RT - PCR. Detailed Embodiments

[0046] The following will further illustrate the essential content and beneficial effects of the present invention in combination with examples. These examples are only used to illustrate the present invention and not to limit it.

[0047] Example 1 Reagents and Instruments

[0048] Experimental animals: Male mice of C57 / BL21 strain at 6 days old after birth.

[0049] The instruments and consumables used in the experiment are as shown in Table 1 below.

[0050] Table 1 Instruments and Reagents

[0051]

[0052]

[0053] Prepare embryonic fibroblast (MEF) medium: DMEM basal medium + 10% FBS + 2 mM GlutaMAX + 1x non - essential amino acids NEAA + 1x penicillin - streptomycin.

[0054] Prepare spermatogonial stem cell medium: StemPro34 SFM base + 1x StemPro34 nutrient supplement + 5 mg / mL bovine serum albumin BSA + 6 mg / mL glucose + 2 mM GlutaMAX + 1x non-essential amino acids NEAA + 1x penicillin-streptomycin double antibody + 1x MEM vitamins + 10 μg / mL d-biotin + 30 μg / mL sodium pyruvate + 0.06% lactic acid + 100 μM ascorbic acid + 30 nM sodium selenite + 60 μM putrescine + 100 μg / mL transferrin + 60 ng / mL progesterone + 30 ng / mL β-estradiol + 10 μM β-mercaptoethanol + 1% FBS + 20 ng / mL EGF + 20 ng / mL GDNF + 10 ng / mL bFGF + 1x103 U / mL LIF.

[0055] Example 2 Establishment of spermatogonial stem cell (SSCs) line from a single mouse

[0056] 1. Isolate mouse testes: After sacrificing the mouse by cervical dislocation, disinfect the mouse body surface with 75% alcohol. Open the abdomen and take out the testis tissue, and place it in a 35 mm culture dish containing pre-cooled D-Hanks buffer with 1% double antibody. After collecting all the testes, wash them 5-6 times repeatedly with D-Hanks buffer to remove blood cells. After the last wash, remove the tunica albuginea on the surface of the testes, and then place the remaining tissue in a 35 mm culture dish with a small amount of buffer. Cut the testes into pieces with scissors.

[0057] 2. Collagenase digestion: Then transfer the testis tissue to a 15 mL centrifuge tube containing 1 mg / mL collagenase, seal it with a sealing film, and shake it in a 37 °C water bath for about 15 min while holding the 15 mL centrifuge tube. Observe the aggregation of the testis tissue every 3 min or so. If it aggregates into a ball, gently shake the centrifuge tube to disperse it and then continue digestion.

[0058] 3. Trypsin digestion: After collagenase digestion, centrifuge at 1000 rpm for 5 min, discard the supernatant, then add 3 mL of 0.05% trypsin to the 15 mL centrifuge tube, seal it with a sealing film, and shake it in a 37 °C water bath for about 5 min while holding the 15 mL centrifuge tube. Then add 10% FBS or 5-8 mL of MEF medium containing 10% FBS to terminate the digestion.

[0059] 4. Purification of SSCs by differential adherence method: After filtering with a 40-μm cell sieve, the filtrate was collected in a 50-mL centrifuge tube, centrifuged at 1000 rpm for 5 min, the supernatant was discarded, and then the cell pellet was resuspended with 1 mL of spermatogonial stem cell medium. Then the cell suspension was inoculated into one well of a 12-well culture plate and placed in an incubator for overnight differential adherence. The next day, the bottom of the culture plate was gently pipetted, the supernatant was collected and transferred to one well of another new 12-well culture plate, and cultured in spermatogonial stem cell medium. The medium was changed by half every 3 days.

[0060] 5. Subculture of spermatogonial stem cells (SSCs): The first subculture was carried out around 15 - 30 days of culture, depending on the growth status of SSCs clone clusters. At the first subculture, the SSCs obtained in step 4 should be cultured until the density of the clone clusters is greater than 60%, preferably greater than 80%, more preferably greater than 90%. The inventors unexpectedly found that the selection of the first subculture time is the key to determining whether SSCs can be successfully established. Subculture at a density of SSCs clone clusters lower than the aforementioned level will result in the inability of the cells after subculture to proliferate.

[0061] The inventors found that when SSCs are subcultured for 1 - 3 generations, there is no need to additionally use feeder cells, otherwise it has a negative effect on the growth of SSCs. The reason may be that there are more somatic cells, and the somatic cells contained in the cell suspension obtained by trypsin digestion can maintain the normal proliferation of SSCs. Starting from the 4th subculture, embryonic fibroblast (MEF) is used as the feeder layer of SSCs to maintain its rapid expansion.

[0062] Preparation of embryonic fibroblast (MEF) feeder layer:

[0063] One day before the 4th and subsequent subcultures, the feeder cells treated with mitomycin (10 μg / mL) were inoculated into a 12-well culture plate, and the density of the feeder cells was above 90%. According to the inventors' experiments, it was found that too sparse feeder layer would lead to the failure of amplification.

[0064] The subculture includes treating (digesting) the cells in the culture plate with trypsin, removing somatic cells from the detached cells after treatment by differential adherence method, and then resuspending the collected cells. In one embodiment, trypsin digestion includes: adding 3 mL of 0.05% trypsin, sealing with a sealing film, in a 37°C water bath, holding a 15-mL centrifuge tube and shaking for digestion for about 5 min, and then adding 10% FBS (or 5 - 8 mL of MEF medium containing 10% FBS) to terminate digestion. After trypsin digestion, differential adherence was carried out in a 12-well plate for 2 h to remove most testicular somatic cells. After collecting the supernatant, centrifugation was carried out at 1000 rpm for 5 min, the supernatant was discarded, and then resuspended with SSCs medium and inoculated on the MEF feeder layer for normal culture, and the medium was changed every two days.

[0065] The representative cell morphology after subculture of spermatogonial stem cells is as Figure 1 shown: Under an optical microscope, the spermatogonial stem cell clone clusters are in the shape of grape clusters, with a smooth outer edge, large cell bodies, about 10 μm in diameter, and relatively large nuclei, which are round or oval; in the early stage of cell culture (P1 generation), the amplification of SSCs is slow. When cultured for 10 - 12 days, only a small number of clone clusters appear. With the increase in the number of passages (above P20, such as P33), the proliferation rate of SSCs significantly accelerates.

[0066] Example 3 Identification and Analysis of Spermatogonial Stem Cell Lines

[0067] Prepare cell smears of SSCs cultured in vitro, and use immunofluorescence staining and RT-PCR methods to detect the expression of SSCs molecular markers (MVH, PLZF, OCT4, and GFRα1) in the cells cultured in vitro.

[0068] 1. Identification by immunofluorescence staining: Discard the culture medium. After rinsing the cells with PBS buffer, add 200 μL of 4% paraformaldehyde solution to each well and fix at room temperature for 30 min. Wash 3 times with PBST, 5 min each time; add 200 μL of 0.5% Triton X-100 solution to each well and permeabilize at room temperature for 20 min (for membrane proteins such as MVH and GFRα1, no permeabilization treatment is required). Wash 3 times with PBST, 5 min each time; add 200 μL of 5% BSA to each well, block at room temperature for 30 min, then add the diluted primary antibody and incubate overnight at 4°C. The next day, discard the primary antibody, wash 3 times with PBST, 5 min each time; add the diluted secondary antibody and incubate in the dark at room temperature for 1 h. Wash 3 times with PBST, 5 min each time; counterstain the cell nuclei with DAPI for 5 - 10 min, wash 3 times with PBST, 5 min each time; add a mounting medium containing a fluorescence quencher and image.

[0069] 2. Identification by RT-PCR: Collect SSCs with 0.05% trypsin digestion solution, centrifuge at 1000 rpm for 5 min, and discard the supernatant. Extract the total RNA in the cells with TRIzol lysis solution, reverse transcribe to synthesize cDNA strands, and then perform RT-PCR detection.

[0070] The results are as Figure 2 (immunofluorescence staining) and Figure 3 shown (RT-PCR) that the cells of the spermatogonial stem cell line prepared by the method of the present invention all express the molecular markers of spermatogonial stem cells, and the localization of the molecular markers is correct: PLZF and OCT4 are localized in the cell nuclei, and MVH and GFRα1 are localized in the cytoplasm and cell membranes.

[0071] The foregoing is a description of the present invention and should not be construed as a limitation thereof. Unless otherwise indicated, the practice of the present invention will employ conventional techniques of organic chemistry, polymer chemistry, biotechnology, etc. Obviously, the present invention can be implemented in other ways than those specifically described in the foregoing description and examples. Other aspects and improvements within the scope of the present invention will be apparent to those skilled in the art to which the present invention pertains. Many changes and variations are possible in accordance with the teachings of the present invention, and thus it is within the scope of the present invention.

Claims

1. A method for preparing a mammalian spermatogonial stem cell line, comprising the following steps: Step 1: Purify and culture spermatogonial stem cells from testis; Step 2: Subculturing the spermatogonial stem cells obtained in step 1 for 1 to 3 generations; Step 3: Subculture the spermatogonial stem cells obtained in step 2 in a feeder layer. Preferably, the feeder layer is an embryonic fibroblast feeder layer.

2. The method of claim 1, wherein step 1 comprises the following steps: (1) Isolating mammalian testicles; (2) digesting the testicular tissue fragments with collagenase, for example, digesting in 1 mg / mL collagenase for about 10-20 minutes; (3) digesting with trypsin, for example, digesting with 0.05% trypsin for about 2-8 minutes after collagenase digestion, and then terminating the digestion; (4) Purification: Somatic cells are removed by differential adhesion method to obtain spermatogonial stem cells, which are then cultured in spermatogonial stem cell culture medium.

3. The method according to claim 1 or 2, wherein the spermatogonial stem cells purified in step 1 are cultured for 15-30 days before being subcultured in step 2.

4. The method according to claim 1 or 2, wherein the spermatogonial stem cells purified in step 1 are cultured until the clone density is greater than 60%, preferably greater than 80%, and more preferably greater than 90%, and then subcultured in step 2.

5. The method according to claim 1 or 2, wherein the subculture in step 2 is performed for 1-3 generations, preferably, for 3 generations.

6. The method according to claim 1 or 2, wherein the subculture in step 3 is carried out for more than 20 generations, preferably for more than 30 generations.

7. The method according to claim 1 or 2, wherein the feeder cell density of the feeder layer is greater than 80%, preferably greater than 90%, more preferably greater than 95%.

8. The method according to any one of claims 1 to 7, wherein the spermatogonial stem cells are cultured and subcultured in a spermatogonial stem cell culture medium, wherein the spermatogonial stem cell culture medium comprises a basal culture medium, nutrients and growth factors, and preferably, the spermatogonial stem cell culture medium does not contain insulin.

9. The method according to claim 1, wherein the spermatogonial stem cell culture medium comprises: basic culture medium StemPro34 SFM base + 1x StemPro34 nutrient supplement + 5mg / mL bovine serum albumin BSA + 6mg / mL glucose + 2mM GlutaMAX + 1x non-essential amino acid NEAA + 1x penicillin-streptomycin double antibody + 1x MEM vitamins + 10μg / mL biotin d-Biotin + 30μg / mL sodium pyruvate + 0.06% lactic acid + 100μM ascorbic acid + 30nM sodium selenite + 60μM putrescine + 100μg / mL transferrin + 60ng / mL progesterone + 30ng / mL β-estradiol + 10μM β-mercaptoethanol + 1% FBS + 20ng / mL EGF + 20ng / mL GDNF + 10ng / mL bFGF + 1x10 3 U / mL LIF.

10. A mammalian spermatogonial stem cell line obtained according to the method of any one of claims 1 to 9.

Images