Culture solution for promoting in-vitro human spermatogenesis and use thereof

Abstract

Provided in the present invention is a culture solution for promoting in-vitro human spermatogenesis and the use thereof. The culture solution comprises the following components in volume fraction: 80%-95% of αMEM, and 5%-20% of a serum substitute and 50-100 ng / mL transforming growth factor β family activin A, 10-50 ng / mL stem cell growth factor, 10-50 ng / mL bone morphogenetic protein, 10-7-10-5 M retinoic acid, 10-50 mM testosterone, 100-200 ng / mL follicle-stimulating hormone, and 50-100 mg / mL pituitary extract. Testicular tissue with meiotic arrest of human germ cells is separated into small pieces, and same are cultured for 10-30 days in the culture solution for promoting in-vitro human spermatogenesis, such that human spermatids can be obtained. The culture solution of the present invention promotes the in-vitro maturation of human male germ cells, which provides a basis for research on human spermatogenesis, and provides technical support for the clinical treatment of male infertility.

Description

A culture medium for promoting human spermatogenesis in vitro and its application Technical Field

[0001] The present invention belongs to the field of biomedicine, and in particular relates to a culture medium for promoting human spermatogenesis in vitro and its application. Background Art

[0002] Spermatogenesis is fundamental to male fertility. It refers to the continuous and complex biological process from spermatogonia to mature sperm, encompassing three stages: spermatogonia self-renewal, spermatocyte meiosis, and spermatogenesis. Spermatogenesis is susceptible to genetic mutations, endocrine disorders, and environmental endocrine disruptors, leading to male infertility.

[0003] Male infertility accounts for approximately 50% of human infertility, 20% of which is due to azoospermia. Approximately 8-12% of patients with azoospermia experience arrested germ cell maturation. Furthermore, the incidence of childhood cancer ranges from 1.0 to 2.5 cases per 1,000 children, and approximately one-third of males are at risk of azoospermia after treatment. Therefore, there is an urgent need to establish a "gold standard" in vitro meiotic culture method for human male germ cells to transform immature testicular germ cells into spermatocytes.

[0004] The establishment of an in vitro human testicular spermatogenesis method provides a research model for studying the genetic, epigenetic and environmental factors in the formation and development of germ cells and testicular somatic cells, as well as the pathological mechanisms of various spermatogenesis disorders, and provides technical reserves for the establishment of fertility preservation in patients with germ cell maturation arrest and prepubertal male tumor patients.

[0005] Summary of the Invention

[0006] The present invention aims to provide a culture medium and its application for promoting human spermatogenesis in vitro, and to establish an in vitro culture system for testicular tissue from patients with clinical spermatogenic disorders. Specifically targeting spermatogonia or spermatocyte maturation arrest, the system repairs spermatogenic disorders through in vitro culture, completes meiosis, and obtains functional haploid sperm. Morphological analysis, multi-omics sequencing, genetic analysis, and functional validation are used to detect haploid spermatids induced in vitro.

[0007] In order to achieve the above object, the present invention provides the following technical solutions:

[0008] In a first aspect, the present invention provides a culture medium for promoting human spermatogenesis in vitro, wherein the culture medium comprises, by volume fraction: 80-95% αMEM and 5-20% serum replacement, as well as 50-100 ng / mL transforming growth factor β family activin A, 10-50 ng / mL stem cell growth factor, 10-50 ng / mL bone morphogenetic protein, 10 -7-10 -5 M retinoic acid, 10-50mM testosterone, 100-200ng / mL follicle-stimulating hormone, and 50-100mg / mL pituitary extract.

[0009] In a second aspect, the present invention provides a culture method for promoting human spermatogenesis in vitro, using the above-mentioned culture medium, comprising the following steps:

[0010] The human testicular tissue with arrested germ cell maturation is separated into small pieces, placed in a culture dish containing the culture medium, and cultured at 32-37° C. in an environment of 4-5% carbon dioxide for 10-30 days, for example, at 32-37° C. in an environment of 4-5% carbon dioxide for 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days, or values ​​and ranges between any two of these values.

[0011] In some embodiments, in the above-mentioned culture method for promoting human spermatogenesis in vitro, the human testis with arrested germ cell maturation is a human testis with arrested meiosis.

[0012] In a third aspect, the present invention provides use of the above-mentioned culture medium in promoting human spermatogenesis in vitro.

[0013] In a fourth aspect, the present invention provides use of the above-mentioned culture medium in promoting spermatogenesis in human testicles with arrested germ cell maturation in vitro.

[0014] In some embodiments, in the use of the above culture medium for promoting spermatogenesis in human testis with arrested germ cell maturation in vitro, the human testis with arrested germ cell maturation is human testis with arrested meiosis.

[0015] In a fifth aspect, the present invention provides a kit comprising the above-mentioned culture medium and optionally instructions for use, wherein the instructions describe information such as the operating steps for culturing cells using the culture medium.

[0016] In a sixth aspect, the present invention provides use of retinoic acid in promoting human spermatogenesis, such as use of retinoic acid in promoting human spermatogenesis in vitro or in vivo.

[0017] In a seventh aspect, the present invention provides use of retinoic acid in promoting spermatogenesis in human testes with arrested germ cell maturation, such as use of retinoic acid in promoting spermatogenesis in human testes with arrested germ cell maturation in vivo or in vitro.

[0018] In some embodiments, the use of the retinoic acid for promoting spermatogenesis in a human testis with arrested germ cell maturation is a human testis with arrested meiosis.

[0019] The present invention has the beneficial effects of culturing meiotically arrested human testicular tissue. By adding various cytokines and hormones to the culture medium, the testicular microenvironment is modulated, promoting the establishment of effective partner-receptor relationships between testicular germ cells and somatic cells, inducing testicular regeneration and obtaining functional gametes. This culture medium provides technical possibilities for in vitro male sperm maturation and a platform for the development of male infertility treatments.

[0020] This culture medium can promote the completion of meiosis of maturation-arrested germ cells and obtain spermatids. In addition, spermatogonia and spermatocytes continue to exist after in vitro culture, indicating that this system can induce the production of spermatids multiple times.

[0021] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention and implement it according to the contents of the specification, the following is a detailed description of the preferred embodiments of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 shows the identification results of human testes with meiotic arrest before culture: A. Immunofluorescence staining of specific proteins of each cell type after testicular tissue sectioning; B is a flow cytometry (FACS) analysis of clinical testicular tissue before culture, 1C: 1x DNA content; 2C: 2x DNA content; 4C: 4x DNA content.

[0023] FIG2 is an immunofluorescence staining diagram of specific proteins of each cell type after 10 days of in vitro culture in Comparative Example 1.

[0024] Figure 3 shows the results of in vitro culture in Example 1, wherein A is an immunofluorescence staining image of specific proteins of each cell type after 10 days of culture; B is a flow cytometry ploidy analysis image after 18 days of culture.

[0025] FIG4 is a diagram of sperm cells induced and obtained in Example 1, and the identification of the sperm cells after drop-smearing by PNA and cell nucleus Hoechst staining.

[0026] FIG5 is a CNV sequencing diagram of the sperm cell genome induced in Example 1.

[0027] Figure 6 shows the results of in vitro culture at different concentrations in Example 2, wherein A is a flow cytometry analysis of the low-concentration groups of cytokines and hormones after 18 days of culture; and B is a flow cytometry analysis of the high-concentration groups of cytokines and hormones after 18 days of culture.

[0028] FIG7 is a flow cytometry ploidy analysis diagram after long-term in vitro culture in Example 3.

[0029] NOA in the figure stands for non-obstructive azoospermia. DETAILED DESCRIPTION

[0030] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

[0031] The present invention discloses a culture medium for promoting in vitro human spermatogenesis and its application. The reagents, instruments, cell lines and the like used in the culture medium for promoting in vitro sperm maturation provided by the present invention can all be purchased on the market.

[0032] Human testicular tissue that has undergone clinical meiotic arrest was collected and cultured in vitro in three dimensions to reconstruct the spermatogenesis process at different stages of germ cells. Culture conditions were further refined based on the type of maturation arrest. Immunofluorescence staining of tissue explants at key time points was performed to identify spermatogonia, spermatocytes, spermatids, and somatic cell-specific marker proteins, such as PLZF, SYCP3, PNA, and SOX9, to assess the status of germ cells and somatic cells in the cultured testicular tissue. Flow cytometry (FACS) was used to analyze the DNA content of cells at different key time points in the cultured testicular tissue, and to examine the production of spermatocytes and haploid spermatids during the in vitro culture of testicular tissue. Haploid cells obtained by FACS were further characterized using methods such as fluorescence staining and CNV sequencing to determine the genetic and epigenetic status of haploid spermatids.

[0033] Preparation before cultivation

[0034] (I) Identification of cell types in human testes with meiotic arrest

[0035] Tissue fixation method: Take about 2mm of meiotically arrested human testicular tissue and fix it with 4% PFA at room temperature for 1 hour. Then transfer it to graded alcohol for gradual dehydration: 70%, 80%, 90% and 100% for 40 minutes each, 100% alcohol: xylene (1:1) for 6 minutes, xylene for 20 seconds, and then the tissue is immersed in wax and embedded in sections.

[0036] Various cell types in meiotically arrested human testes were immunostained with specific proteins, including DDX4 for germ cells, SOX9 for Sertoli cells, PLZF for spermatogonia, SYCP3 for spermatocytes, PNA for spermatids, and Hoechst 33342 for nuclear DNA.

[0037] The results are shown in Figure 1. As can be seen from Figure 1A, immunofluorescence staining showed that the specimen expressed the germ cell marker protein DDX4, spermatogonia marker protein PLZF, and spermatocyte marker protein SYCP3, but did not express the spermatid marker PNA, indicating that the germ cells of this tissue were arrested at the spermatocyte stage.

[0038] As can be seen from B in Figure 1, no haploid cells are visible at this time, while the tetraploid ratio is as high as 15%, indicating that spermatocytes but no spermatids exist in this specimen, and the results are consistent with the immunofluorescence results.

[0039] Example 1

[0040] The human testis with meiotic arrest is induced and cultured in the culture medium of the present invention.

[0041] Method for promoting in vitro human spermatogenesis: cut meiotically arrested human testicular tissue into 3 mm-sized pieces, place them in a culture dish, add 1 mL of culture medium and culture at 34° C. and 5% CO 2 .

[0042] The specific composition of the culture medium used in the experiment is as follows:

[0043] 90% (v / v) αMEM, 10% (v / v) serum replacement (KSR), 100 ng / mL transforming growth factor β family activin A (Activin A, R&D Systems), 20 ng / mL stem cell growth factor (SCF, R&D Systems), 20 ng / mL bone morphogenetic protein (BMP 4 / 7, R&D Systems), 10 -6 M retinoic acid (RA), 10 mM testosterone (Acros Organics), 200 ng / mL follicle-stimulating hormone (FSH, Sigma), and 50 mg / mL pituitary extract (BPE, Corning Life Sciences).

[0044] After 10 days of tissue culture, the tissue was collected, fixed in 4% PFA, and stained with PNA and SYCP3. The results are shown in Figure 3A. After 10 days of in vitro induction, PNA-positive cells, namely spermatids, appeared in the testicular tissue, indicating that the optimized induction system can promote meiotically arrested spermatocytes to complete meiosis and obtain spermatids. In addition, spermatocytes still exist after in vitro culture, indicating that the system can induce the production of spermatids multiple times.

[0045] Tissue cultured in optimized medium for 18 days was subjected to flow cytometry sorting for ploidy. The results are shown in Figure 3B, where 1C represents 1x DNA content; 2C represents 2x DNA content; and 4C represents 4x DNA content. At this point, approximately 5% haploid cells were obtained, and the purified haploid cells were collected for subsequent validation experiments.

[0046] Comparative Example 1

[0047] Meiotically arrested human testes were cultured in conventional culture medium

[0048] Method for promoting spermatogenesis in vitro: cut meiotically arrested human testicular tissue into 3 mm-sized pieces, place them in a culture dish, add 1 mL of conventional culture medium and culture at 34° C. and 5% CO 2 .

[0049] The specific components of the conventional culture medium used in the experiment are as follows:

[0050] 90% (v / v) αMEM, 10% (v / v) serum replacement (KSR), 100 ng / mL transforming growth factor beta family activin A (Activin A, R&D Systems), 20 ng / mL stem cell growth factor (SCF, R&D Systems), 20 ng / mL bone morphogenetic protein (BMP 4 / 7, R&D Systems), 20 ng / mL basic fibroblast growth factor (bFGF, R&D Systems), 20 ng / mL epidermal growth factor (EGF, R&D Systems), 10 mM testosterone (Acros Organics), 200 ng / mL follicle-stimulating hormone (FSH, Sigma) and 50 mg / mL pituitary extract (BPE, Corning Life Sciences).

[0051] Compared with Example 1, Comparative Example 1 added 20 ng / mL basic fibroblast growth factor (bFGF, R&D Systems) and 20 ng / mL epidermal growth factor (EGF, R&D Systems), but lacked retinoic acid.

[0052] After 10 days of culture, tissue samples were harvested, fixed in 4% PFA, and stained with PLZF and SYCP3. The results, shown in Figure 2, show that after 10 days of in vitro induction with conventional culture medium, spermatogonia proliferated significantly, while spermatocytes were no longer visible. This suggests that the microenvironment provided by this induction system promotes spermatogonia proliferation and spermatocyte apoptosis within the testicular tissue, failing to achieve the desired in vitro spermatogenesis. This suggests that the removal of basic fibroblast growth factor and epidermal growth factor can prevent the excessive proliferation of spermatogonia and the inhibition of meiosis within the seminiferous tubules, while the addition of retinoic acid can promote the initiation and completion of meiosis, resulting in a more optimal induction of spermatogenesis in vitro.

[0053] Post-culture testing

[0054] The testicular tissue in Example 1 was induced in vitro to obtain sperm cell specific markers for detection

[0055] Flow cytometry was used to purify haploid cells and prepare cell suspension droplets. The specific steps are as follows:

[0056] Testicular tissue cultured for 18 days was harvested under a stereoscope and placed in a 3.5-cm culture dish. A two-step enzymatic digestion method was used to prepare a single-cell suspension, followed by Hoechst 33342 nuclei staining at 37°C for 20 minutes. Flow cytometry was then performed for ploidy sorting, with the proportion of 1C cells being approximately 5%. Cells with a DNA content of 1C were collected, the collection tube was centrifuged, the supernatant discarded, and the pellet resuspended in 50 μl of culture medium before being mounted on a slide.

[0057] The results are shown in FIG4 , where the cells were plated and identified by PNA and cell nucleus Hoechst staining.

[0058] Identification of genetic characteristics of sperm cells obtained by in vitro induction of testicular tissue in Example 1

[0059] Flow cytometry purification of haploid cells, the specific steps are as follows:

[0060] Testicular tissue cultured for 18 days was harvested under a stereoscope and placed in a 3.5-cm culture dish. A two-step enzymatic digestion method was used to prepare a single-cell suspension, followed by Hoechst 33342 nuclei staining at 37°C for 20 minutes. Flow cytometry was then performed for ploidy sorting, and cells with a DNA content of 1C were collected. The cell suspension was then spread flat in a culture dish for single-cell selection.

[0061] Microscopically selected single-cell sequencing:

[0062] Single cells were selected under a microscope and placed in a single tube of sterile, enzyme-free PCR. Single-cell amplification and library construction were then performed, and CNV sequencing was further used to verify the chromosome integrity of the obtained haploid cells. As shown in Figure 5, in vitro induction can obtain single sperm cells with complete X and Y genomes.

[0063] Example 2

[0064] The human testis with meiotic arrest was cultured in the culture medium of the present invention at different concentrations.

[0065] Method for promoting human spermatogenesis in vitro: human testicular tissue with meiotic arrest was divided into 3 mm tissue blocks, placed in a culture dish, and divided into a low-concentration group and a high-concentration group. 1 mL of culture medium was added and cultured at 34°C and 5% CO2.

[0066] The specific composition of the culture medium used in the experiment is as follows:

[0067] Low concentration group: 95% (v / v) αMEM, 5% (v / v) serum replacement (KSR), 50ng / mL transforming growth factor β family activin A (Activin A, R&D Systems), 10ng / mL stem cell growth factor (SCF, R&D Systems), 10ng / mL bone morphogenetic protein (BMP 4 / 7, R&D Systems), 10 -7 M retinoic acid (RA), 10 mM testosterone (Acros Organics), 100 ng / mL follicle-stimulating hormone (FSH, Sigma), and 50 mg / mL pituitary extract (BPE, Corning Life Sciences);

[0068] High concentration group: 80% (v / v) αMEM, 20% (v / v) serum replacement (KSR), 100ng / mL transforming growth factor β family activin A (Activin A, R&D Systems), 50ng / mL stem cell growth factor (SCF, R&D Systems), 50ng / mL bone morphogenetic protein (BMP 4 / 7, R&D Systems), 10 -5 M retinoic acid (RA), 50 mM testosterone (Acros Organics), 200 ng / mL follicle-stimulating hormone (FSH, Sigma), and 100 mg / mL pituitary extract (BPE, Corning Life Sciences).

[0069] Post-culture testing

[0070] The testicular tissue induced in vitro at different concentrations in Example 2 was subjected to flow cytometry ploidy analysis.

[0071] Prepare tissue blocks into cell suspensions for ploidy analysis. The specific steps are as follows:

[0072] Under a stereomicroscope, two groups of testicular tissue cultured for 18 days at different concentrations were selected and placed in 3.5 cm culture dishes. Single-cell suspensions were prepared using a two-step enzymatic digestion method, followed by Hoechst 33342 nuclear staining at 37°C for 20 minutes. Flow cytometry analysis of ploidy was performed, and the results are shown in Figure 6. The results showed that human testicular tissue with meiotic arrest was able to complete spermatogenesis in both the low- and high-concentration culture medium groups described herein, yielding 7-8% spermatids.

[0073] Example 3

[0074] The human testis with meiotic arrest is cultured for a long time in the culture medium of the present invention.

[0075] Method for promoting in vitro human spermatogenesis: cut meiotically arrested human testicular tissue into 3 mm-sized pieces, place them in a culture dish, add 1 mL of culture medium and culture at 34° C. and 5% CO 2 .

[0076] The specific composition of the culture medium used in the experiment is as follows:

[0077] 90% (v / v) αMEM, 10% (v / v) serum replacement (KSR), 100 ng / mL transforming growth factor β family activin A (Activin A, R&D Systems), 20 ng / mL stem cell growth factor (SCF, R&D Systems), 20 ng / mL bone morphogenetic protein (BMP 4 / 7, R&D Systems), 10 -6 M retinoic acid (RA), 10 mM testosterone (Acros Organics), 200 ng / mL follicle-stimulating hormone (FSH, Sigma), and 50 mg / mL pituitary extract (BPE, Corning Life Sciences).

[0078] Post-culture testing

[0079] The long-term cultured testicular tissue in Example 3 was subjected to flow cytometry ploidy analysis.

[0080] Prepare tissue blocks into cell suspensions for ploidy analysis. The specific steps are as follows:

[0081] Testicular tissue cultured for 25 days was harvested under a stereomicroscope and placed in a 3.5 cm culture dish. A single-cell suspension was prepared using a two-step enzymatic digestion method, followed by Hoechst 33342 nuclear staining at 37°C for 20 minutes. Flow cytometry analysis of ploidy was performed, and the results are shown in Figure 7. The results show that meiotically arrested human testicular tissue can be cultured for up to 25 days in the culture medium described herein and yield induced spermatids at a rate of approximately 5%.

[0082] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0083] The above-described embodiments merely illustrate several implementations of the present invention. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that variations and modifications are possible within the scope of the present invention, as would be apparent to one skilled in the art. These variations and modifications fall within the scope of the present invention. Therefore, the scope of the present invention is governed by the appended claims.

Claims

1. A culture medium for promoting human spermatogenesis in vitro, characterized in that: The culture medium comprises, by volume fraction, 80-95% αMEM and 5-20% serum substitute, as well as 50-100 ng / mL transforming growth factor β family activin A, 10-50 ng / mL stem cell growth factor, 10-50 ng / mL bone morphogenetic protein, 10 -7 -10 -5 M retinoic acid, 10-50mM testosterone, 100-200ng / mL follicle stimulating hormone and 50-100mg / mL pituitary extract.

2. A method for promoting in vitro human spermatogenesis, characterized in that: Using the culture solution described in claim 1, comprising the following steps: The human testicular tissue with blocked germ cell maturation is separated into small pieces, placed in a culture dish containing the culture medium, and cultured at 32-37° C. and 4-5% carbon dioxide environment for 10-30 days.

3. The method for promoting in vitro human spermatogenesis according to claim 2, characterized in that: The germ cell maturation arrested human testis is a meiotically arrested human testis.

4. Use of the culture medium according to claim 1 in promoting human spermatogenesis in vitro.

5. Use of the culture medium according to claim 1 in promoting spermatogenesis in human testicles with blocked germ cell maturation in vitro.

6. The use according to claim 5, characterized in that The germ cell maturation arrested human testis is a meiotically arrested human testis.

7. A kit comprising the culture solution according to claim 1.

8. Use of retinoic acid in promoting human spermatogenesis.

9. Use of retinoic acid in promoting spermatogenesis in human testes with arrested germ cell maturation.

10. The use according to claim 9, characterized in that The germ cell maturation arrested human testis is a meiotically arrested human testis.

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