System and method for differentiating spermatogonium in vitro

Abstract

The invention provides a method for differentiating spermatogonial stem cells and / or spermatogonial progenitor cells of mammals to obtain spermatogonial cells. The invention also provides the spermatogonial cells prepared by the method. The invention also provides a culture medium for promoting spermatogonial stem cells and / or spermatogonial progenitor cells of mammals to differentiate into spermatogonial cells.

Description

Technical Field

[0001] The present invention relates to the field of germ cell research. Specifically, the present invention provides a method and system for in vitro differentiation of spermatogonia. Background Art

[0002] Differentiation of stem cells is the process by which an unspecialized ("uncommitted") or less specialized cell acquires the characteristics of a specialized cell (e.g., a spermatogonia). A differentiated cell is a cell that has taken on a more specialized ("committed") position within the lineage of cells. The term "committed" when applied to the differentiation process refers to a cell that has progressed to such a point in the differentiation pathway that, under normal circumstances, it will continue to differentiate into a specific cell type or subset of cell types and, under normal circumstances, cannot differentiate into another cell type or regress to a less differentiated cell type.

[0003] Spermatogonial stem cells (SSCs) are undifferentiated germ cells located in the basal layer of the seminiferous tubules of the testis, which have the dual ability of self-renewal and directed differentiation into sperm. As the core cell population in the development of the male reproductive system, SSCs maintain the homeostasis of the stem cell pool through asymmetric division, while generating differentiated spermatogonial progenitor cells (SPCs), and finally forming mature gametes through meiosis.

[0004] The positive molecular markers of spermatogonial stem cells include: GFRα1, PLZF (ZBTB16), NANOS2, RET, etc., and they do not express differentiation-related genes (such as KIT, STRA8). Spermatogonial stem cells rely on GDNF (glial cell-derived neurotrophic factor) secreted by supporting cells (such as Sertoli cells) in the niche to maintain stemness, and do not directly respond to retinoic acid (RA) signals.

[0005] Spermatogonial progenitor cells are transitional germ cells differentiated from spermatogonial stem cells. They lose their self-renewal ability but have the potential to differentiate into spermatocytes. Under the stimulation of RA and other signals, they initiate the meiotic program and differentiate into primary spermatocytes. Positive molecular markers of spermatogonial progenitor cells include KIT (CD117), STRA8, SYCP3 (meiotic initiation marker), etc. Spermatogonial progenitor cells express RA receptors (RARα, RARγ), directly respond to retinoic acid signals, and trigger the expression of meiotic genes (such as Stra8, Rec8).

[0006] This field also needs more extensive research on the stemness and transformation mechanisms of mammalian spermatogonial stem cells and spermatogonia, and the development of better methods and systems for in vitro differentiation of spermatogonia. Summary of the invention

[0007] The present application provides a novel method and system for differentiating mammalian cells to obtain spermatogonia. The method of the present invention can be used to differentiate spermatogonia from spermatogonial stem cells and / or spermatogonial progenitor cells of mammals.

[0008] Specifically, the present invention provides a method for differentiating mammalian cells to obtain spermatogonia, comprising culturing spermatogonia in a differentiation medium. In the method of the present invention, the differentiation medium contains retinoic acid (RA). In one aspect of the present invention, the content of retinoic acid is 0.2-5 μM, preferably about 1 μM.

[0009] In one aspect of the present invention, the culturing time in the differentiation medium is no more than 32 hours, preferably no more than 24 hours, for example 6-24 hours.

[0010] In another aspect of the present invention, a spermatogonial differentiation medium is provided.

[0011] The spermatogonial differentiation medium provided by the present invention for the aforementioned differentiation method comprises various basal medium or nutrient medium suitable for culturing cells. The basal medium or nutrient medium contains a cell culture medium containing nutrients that promote cell proliferation, generally containing isotonic saline, buffer, 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), KODMEM (Knockout DMEM), F12 / 50% DMEM, etc.

[0012] Substances that can be used to maintain cell growth in the differentiation medium of the present invention include putrescine, progesterone and β-mercaptoethanol, etc. In one aspect of the present invention, the differentiation medium contains fetal bovine serum (FBS). In one aspect of the present invention, the stem cell culture medium in the above method contains vitamins.

[0013] 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 / glutamate tyrosine, proline, and serine.

[0014] In one aspect of the present invention, the stem cell culture medium in the aforementioned method further contains antibiotics.

[0015] In one aspect of the present invention, the method comprises the step of culturing mammalian spermatogonial stem cells into spermatogonial progenitor cells.

[0016] In one aspect of the present invention, the mammalian spermatogonial stem cells are cultured to spermatogonial progenitor cells, comprising:

[0017] Step 1: Purify and culture spermatogonial stem cells from mammalian testes;

[0018] Step 2: Subculturing the spermatogonial stem cells obtained in step 1 for 1 to 3 generations;

[0019] Step 3: Subculture the spermatogonial stem cells obtained in step 2 in a feeder layer (e.g., an embryonic fibroblast feeder layer) to obtain a spermatogonial stem cell line after multiple generations of subculture.

[0020] In one aspect of the present invention, step 1 comprises the following steps:

[0021] (1) Collagenase digestion of testicular tissue;

[0022] (2) Trypsin digestion;

[0023] (3) Purification: remove somatic cells and obtain spermatogonial stem cells;

[0024] (4) The obtained spermatogonial stem cells are cultured in a spermatogonial stem cell culture medium.

[0025] In one aspect of the present invention, step 1 in the aforementioned method comprises the following steps:

[0026] (1) Testis isolation: isolating and obtaining testicles from mammals such as mice;

[0027] (2) collagenase digestion, for example, digestion in 1 mg / mL collagenase for about 10-20 minutes;

[0028] (3) trypsin digestion, for example, digestion with 0.05% trypsin for about 2-8 minutes after collagenase digestion, and then terminating the digestion;

[0029] (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.

[0030] In one aspect of the present invention, the spermatogonial stem cells purified in step 1 of the aforementioned method are cultured for 15-30 days before being subcultured in step 2.

[0031] In one aspect of the present invention, the subculture in step 2 of the aforementioned method is carried out for 1-3 generations, preferably, for 3 generations.

[0032] In one aspect of the present invention, the subculture in step 3 of the aforementioned method is performed for more than 20 generations, preferably for more than 30 generations.

[0033] In one aspect of the present invention, in the aforementioned method, the density of the feeder layer (eg, embryonic fibroblast feeder layer) is greater than 80%, preferably greater than 90%, and more preferably greater than 95%.

[0034] In the present invention, in the aforementioned method, the spermatogonial stem cell culture medium is a culture medium (or culture fluid) suitable for the culture of stem cells, which includes maintaining the proliferation ability, developmental potential and stemness of stem cells. The stem cell culture medium that can be used in the present invention can be provided and obtained in a commercially available form. The culture medium that can be used in the present invention can be provided and obtained in a commercially available form.

[0035] In one aspect of the present invention, the spermatogonial stem cell culture medium comprises various basal culture mediums or nutrient mediums suitable for culturing cells, particularly stem cell growth. The basal culture medium or nutrient medium contains a cell culture medium containing nutrients that promote cell proliferation, generally containing isotonic saline, buffer, a protein source (in the form of one or more added proteins or amino acids) and other externally added nutrients and growth factors. The basal culture medium or nutrient medium that can be used for the present invention can be provided and obtained in a commercially available form, for example, including StemPro-34SFM, StemPro-34supplement, etc.

[0036] Substances that can be used to promote cell growth in the spermatogonial stem cell culture medium of the present invention include transferrin, sodium pyruvate, progesterone, β-estradiol and β-mercaptoethanol, etc. Growth factors that can be used in the spermatogonial stem cell culture medium of the present invention include cytokines such as epidermal growth factor EGF, glial cell-derived neurotrophic factor GDNF, basic fibroblast growth factor bFGF, leukemia inhibitory factor LIF, etc. It is worth noting that the spermatogonial stem cell culture medium of the present invention does not include insulin or insulin-like growth factor-1. The inventor unexpectedly found that in the method of the present invention for establishing a stable spermatogonial stem cell line, adding insulin to the culture medium for culturing spermatogonial stem cells will lead to the destruction of spermatogonial stem cell growth (typical phenomenon is atrophy of stem cells) and even cause spermatogonial stem cell death.

[0037] In one aspect of the present invention, the spermatogonial stem cell culture medium comprises: StemPro34 SFM base + 1xStemPro34 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.

[0038] The present invention also provides a mammalian spermatogonia. In one embodiment, the mammalian spermatogonia is prepared by the above method. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 The exemplary subculture of spermatogonial stem cells using the method of the present invention is shown.

[0040] Figure 2 The identification and analysis of spermatogonia prepared by the method provided by the present invention using RT-PCR are shown: changes in spermatogonia maintenance and differentiation-related mRNAs during the differentiation process.

[0041] Figure 3 The identification and analysis of spermatogonia prepared by the method provided by the present invention are shown by immunofluorescence staining. DETAILED DESCRIPTION

[0042] The essential contents and beneficial effects of the present invention will be further described below in conjunction with embodiments, which are only used to illustrate the present invention rather than to limit the present invention.

[0043] Example 1 Reagents and Instruments

[0044] Experimental animals: 6-day-old C57 / BL21 male mice.

[0045] The instruments and consumables used in the experiment are listed in Table 1.

[0046] Table 1 Instruments and reagents

[0047]

[0048]

[0049] Prepare embryonic fibroblast (MEF) culture medium: DMEM basal medium + 10% FBS + 2mM GlutaMAX + 1x non-essential amino acid NEAA + 1x penicillin-streptomycin.

[0050] Prepare spermatogonial stem cell culture medium: StemPro34 SFM base + 1x StemPro34 nutrients 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 + 1x103U / mL LIF.

[0051] Prepare spermatogonial differentiation medium: Minimum Essential medium Alpha + 2mM GlutaMAX + 1×NEAA + 1x penicillin-streptomycin double antibody + 60μM putrescine + 60ng / mL progesterone + 25μg / mL insulin + 10μM β-mercaptoethanol + 1μM retinoic acid (RA) + 10% FBS.

[0052] Example 2 Preparation of mouse spermatogonial stem cells

[0053] 1. Isolation of mouse testicles: After killing mice by cervical dislocation, disinfect the mouse body surface with 75% alcohol, cut open the abdomen and remove the testicular tissue, and place it in a 35mm culture dish containing pre-cooled D-Hanks buffer containing 1% double antibody. After the testicles are collected, wash them repeatedly with D-Hanks buffer for 5-6 times to remove blood cells. After the last wash, remove the white film on the surface of the testicles, and then place the remaining tissue in a 35mm culture dish containing a small amount of buffer, and cut the testicles into pieces with scissors.

[0054] 2. Collagenase digestion: Then transfer the testicular tissue to a 15mL centrifuge tube containing 1mg / mL collagenase, seal it with a sealing film, and shake the 15mL centrifuge tube in a 37℃ water bath for about 15 minutes. Every 3 minutes or so, observe the clumping of the testicular tissue. If it clumps, gently shake the centrifuge tube to disperse it and continue digestion.

[0055] 3. Trypsin digestion: After collagenase digestion, centrifuge at 1000 rpm for 5 minutes, remove the supernatant, and then add 3 mL of 0.05% trypsin to a 15 mL centrifuge tube. After sealing with a sealing film, shake the 15 mL centrifuge tube in a 37°C water bath for about 5 minutes, and then add 10% FBS or 5-8 mL of MEF culture medium containing 10% FBS to terminate the digestion.

[0056] 4. Purify SSCs by differential adhesion method: After filtering with a 40μm cell sieve, collect the filtrate in a 50mL centrifuge tube, centrifuge at 1000rpm for 5min, discard the supernatant, and then resuspend the cell pellet with 1mL spermatogonial stem cell culture medium, and then inoculate the cell suspension in one well of a 12-well culture plate and place it in an incubator for overnight differential adhesion. The next day, gently blow the bottom of the culture plate, collect the supernatant, and transfer it to one well of another new 12-well culture plate and culture it in spermatogonial stem cell culture medium. Change half the volume of the medium every 3 days.

[0057] 5. Spermatogonial stem cells (SSCs) subculture: Starting from the 4th subculture, MEFs are used as the SSCs feeder layer to maintain their rapid expansion.

[0058] One day before the fourth and subsequent subcultures, feeder layer cells treated with mitomycin (10 μg / mL) were seeded in a 12-well culture plate, and the feeder layer cell density was above 90%.

[0059] Representative cell morphology of spermatogonial stem cells after subculture Figure 1 As shown: Under an optical microscope, spermatogonial stem cell clones are grape-like, with smooth peripheral edges, large cell bodies with a diameter of about 10μm, and large, round or oval nuclei; in the early stage of cell culture (P1 generation), SSCs proliferate slowly, and only a small number of clones appear after 10-12 days of culture. As the number of passages increases (above P20, such as P33), the proliferation rate of SSCs accelerates significantly.

[0060] Example 3 Induction of differentiation of mouse spermatogonial stem cells in vitro

[0061] After culturing SSCs for 5 months, wild-type spermatogonial progenitor cells (SPCs) were digested and collected with 0.05% trypsin, and then SPCs were seeded in a 12-well plate coated with 0.2% gelatin for 1 h to remove feeder cells as thoroughly as possible.

[0062] Approximately 10,000 SPCs cells were seeded onto a 24-well plate coated with Matrigel (1:80 dilution). After 24 h, the SSCs cell culture medium was replaced with differentiation medium for culture.

[0063] Example 4 Spermatogonial cell line identification and analysis

[0064] The cells differentiated and cultured in Example 3 were subjected to immunofluorescence staining and RT-PCR to detect the expression of spermatogonia molecular markers.

[0065] qRT-PCR identification: SSCs were collected with 0.05% trypsin digestion solution, centrifuged at 1000rpm for 5min, and the supernatant was discarded. Total RNA in the cells was extracted with TRIzol lysis solution, and reverse transcribed to synthesize cDNA chains. Subsequently, qRT-PCR was performed to detect the expression changes of spermatogonial maintenance and differentiation-related mRNAs during differentiation.

[0066] The results are as follows Figure 2 As shown. Compared with the control group (0h), the expression levels of spermatogonial maintenance-related mRNAs (Oct4 and Gfrα1) began to decrease significantly after 6h of differentiation, and the Plzf mRNA level decreased significantly after 12h. At 24h of differentiation, the expression of the above mRNAs was almost undetectable. On the contrary, the expression levels of spermatogonial differentiation-related mRNAs (Stra8, Kit, Dmrt1 and Ccnd2) increased significantly with the extension of differentiation time.

[0067] The above results indicate that the differentiation method and system provided by the present invention can induce spermatogonia to express differentiation-related mRNAs.

[0068] Immunofluorescence staining identification: discard the culture medium, rinse the cells with PBS buffer, add 200μL 4% paraformaldehyde solution to each well, fix at room temperature for 30min, wash 3 times with PBST, 5min each time; add 200μL 0.5% Triton X-100 solution to each well, permeabilize the membrane for 20min at room temperature, wash 3 times with PBST, 5min each time; add 200μL 5% BSA to each well, block at room temperature for 30min, add diluted primary antibody, and incubate at 4℃ overnight. The next day, discard the primary antibody, wash 3 times with PBST, 5min each time; add diluted secondary antibody, incubate at room temperature in the dark for 1h, wash 3 times with PBST, 5min each time; counterstain the nucleus with DAPI for 5-10min, wash 3 times with PBST, 5min each time; add sealing solution containing fluorescence quencher, and image.

[0069] The results are as follows Figure 3 Co-immunostaining of PLZF, STRA8 and KIT showed that about 95% of undifferentiated SPCs (PLZF + STRA8 - KIT - Cells) enter the differentiation stage after 6 hours of differentiation (PLZF + STRA8 +KIT - cells), which efficiently developed into early differentiated spermatogonia (PLZF) after 12 h of differentiation. - STRA8 + KIT + Even after 24 h of differentiation, no late differentiated spermatogonia were observed (PLZF - STRA8 - KIT + The cells represent mid- to B-type spermatogonia) and pre-leptotene spermatocytes (PLZF - STRA8 + KIT - ).

[0070] These results indicate that the differentiation method and system provided by the present invention can efficiently induce differentiation of wild-type SPCs without entering meiosis.

[0071] The above is an explanation of the present invention and cannot be regarded as a limitation of the present invention. Unless otherwise noted, the practice of the present invention will use the conventional techniques of organic chemistry, polymer chemistry, biotechnology, etc., and it is obvious that the present invention can also be realized in other ways except that it is particularly described in the above description and embodiments. 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 belongs. According to the teachings of the present invention, many changes and variations are feasible, and therefore they are within the scope of the present invention.

Claims

1. A method for differentiating mammalian cells to obtain spermatogonia, comprising culturing spermatogonia in a differentiation medium, wherein the differentiation medium contains retinoic acid (RA), preferably, the content of retinoic acid is 0.2-5 μM, more preferably about 1 μM.

2. The method of claim 1, wherein the culturing time in the differentiation medium is no more than 32 hours, preferably no more than 24 hours, for example 6-24 hours.

3. The method of claim 1 or 2, comprising the step of culturing spermatogonial stem cells to spermatogonial progenitor cells.

4. The method of claim 3, wherein the spermatogonial stem cells are cultured to spermatogonial progenitor cells by the following method: 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: The spermatogonial stem cells obtained in step 2 are subcultured in the feeder layer.

5. The method of claim 4, 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.

6. The method according to claim 4, wherein the subculture in step 2 is carried out for 1-3 generations, preferably, for 3 generations, and the subculture in step 3 is carried out for more than 20 generations, preferably for more than 30 generations.

7. The method according to any one of claims 1-6, wherein the differentiation medium comprises a basal medium, nutrients and growth factors, preferably, the differentiation medium comprises insulin, more preferably, the content of insulin is 5-100 μg / mL, for example, 25 μg / mL.

8. The method according to claim 7, wherein the differentiation medium comprises: Minimum Essential medium Alpha + 2mM GlutaMAX + 1×NEAA + 1x penicillin-streptomycin double antibody + 60μM putrescine + 60ng / mL progesterone + 25μg / mL insulin + 10μM β-mercaptoethanol + 1μM retinoic acid (RA) + 10% FBS.

9. The method according to any one of claims 3 to 6, 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.

10. Mammalian spermatogonia obtained by the method according to any one of claims 1 to 9.

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