CN115074307B - Method for reprogramming fibroblast induced by small molecule compound into testis support cell and application thereof - Google Patents
Method for reprogramming fibroblast induced by small molecule compound into testis support cell and application thereof Download PDFInfo
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Abstract
The present invention provides a method for reprogramming mammalian fibroblasts into testicular support cells (CiSCs), testicular support cells (CiSCs) obtained by the method and uses thereof. The method is easy to operate, does not introduce exogenous genes, is hopeful to be developed into a widely applied method for obtaining the non-testis-source support cells, not only provides a cell model and a theoretical basis for basic research and drug development of reproductive system diseases, but also brings wide prospects for application of testis support cells in the field of regenerative medicine.
Description
Technical Field
The invention relates to the technical fields of cell biology and tissue engineering, in particular to a method for reprogramming fibroblasts into testis support cells by using a small molecular compound and application thereof.
Background
Testis support cells (Sertoli cells) are one of the important somatic cells in testis, are an important component of seminiferous tubules, can provide physical support and stable microenvironment for spermatogenesis, provide nutritional support for spermatogenesis, and have unique immune-free characteristics so as to protect germ cells from body immune attack and foreign matter interference. The Sertoli cell has very wide application in the fields of basic research and clinical treatment, can be used as a model cell for in-vitro reproductive system related mechanism research and reproductive toxicology evaluation, and can be used as a seed cell for treating testicular dysplasia through participating in tissue reconstruction. In addition, the Sertoli cells play an important role in the field of regenerative medicine, and can provide nutrition and immune protection for non-testis cells after transplantation, thereby being beneficial to the survival of transplanted cells and reducing the rejection reaction of organisms. However, mature Sertoli cells are mitotically inactive, and long-term culture in vitro can lead to loss of their functional characteristics, and human Sertoli cells are very limited in availability, making large-scale culture in vitro difficult. Therefore, it is necessary to find an effective strategy to obtain replacement cells for Sertoli cells independent of donor testis cells.
Cell reprogramming is a research hotspot in the fields of tissue engineering and regenerative medicine, and the advent of this technology created new opportunities for drug screening, disease modeling, artificial organ development and cell therapies. There have been a number of studies demonstrating the overexpression of lineage specific transcription factors in somatic cells to induce direct transformation of differentiated cells into target cells, and although these studies open up promising approaches to obtain functional support cells, the risk of genomic insertion of foreign DNA sequences poses safety issues for their future use, which requires elimination of reliance on genetic manipulation. In recent years, more and more research has shown that the role and advantage of small molecules in cell fate reprogramming can overcome the limitations of genetic manipulation. And the small molecule compounds are easy to manufacture, preserve and standardize. However, there has been no report on the conversion of fibroblasts into supporting cells using a chemical reprogramming strategy.
Disclosure of Invention
The invention aims to provide a method for inducing fibroblast to be reprogrammed into testis support cells by using a small molecular compound, which has simple operation and high induction efficiency and provides an effective solution for the problems of insufficient testis support cell sources and the like in clinic.
Specifically, the present invention provides the following:
1. a method of reprogramming/inducing mammalian fibroblasts into testicular-like support cells (CiSCs), the method comprising the steps of:
(1) Treating the isolated mammalian fibroblasts with a BET bromodomain inhibitor; and
(2) The cells from step (1) are then further treated with a glycogen synthase kinase (GSK-3) inhibitor and Retinoic Acid (RA), thereby obtaining the testicular support cells (CiSCs).
The BET bromodomain inhibitors are a class of highly specific and potent BET inhibitors that function by competing binding to the bromodomain and BRD domain of the external terminal (BET) family of proteins. The BET family protein can be specifically combined with an activated chromatin structural domain, so that a cell specific gene expression mode is maintained, and the inhibition of the BET family protein can lead to the inactivation of a fibrosis growth factor receptor, so that a fibrosis transcription factor is inhibited, and the reprogramming of cells is facilitated.
The glycogen synthase kinase (GSK-3) inhibitor is a type of inhibitor which can reduce the targeted ubiquitination degradation of beta-catenin by inhibiting glycogen synthase kinase-3 beta (GSK-3 beta), so as to strengthen the Wnt/beta-catenin signal path, wherein the signal path can participate in regulating and controlling cell self-renewal, embryo development and organogenesis and plays a key role in maintaining pluripotency and in the reprogramming process of somatic cells.
Retinoic Acid (RA) is an agonist of retinoic acid receptor, and can improve the reprogramming Cheng Xiaolv and guide target cells to differentiate germ cells by regulating retinoic acid signal channels to participate in the processes of embryo development, germ system differentiation and the like.
2. The method according to 1 above, wherein the BET bromodomain inhibitor is I-BET151 and/or the glycogen synthase kinase (GSK-3) inhibitor is Riluzole (Riluzole).
3. The method according to 1 above, wherein in step (1) the BET bromodomain inhibitor is added during the logarithmic growth phase of the fibroblasts, the treatment time is preferably about 0.5 hours to 10 days, more preferably about 1-5 days, most preferably about 4 days; and/or wherein said step (2) is performed for about 10 to 40 days, preferably about 25 days.
4. The method according to 1 above, wherein the fibroblast is a primary fibroblast, preferably an embryonic fibroblast, visceral fibroblast or skin fibroblast; and/or the mammal is a human, mouse, rat, monkey, dog, cat, cow, rabbit, horse or pig.
5. The method according to 1 above, wherein the step (1) is performed in DMEM medium containing FBS; and/or said step (2) is performed in a testis-supporting cell induction medium, wherein said testis-supporting cell induction medium is DMEM medium comprising FBS, insulin-like growth factor, epidermal growth factor, fibroblast growth factor 9, follicle stimulating hormone and prostaglandin D2.
6. The method of any one of claims 1-5 above, further comprising:
1) Identifying expression of a testis support cell-related gene in said testis-like support cells (CiSCs), wherein said testis support cell-related gene is selected from the group consisting of Wt1, krt18, fshr, ar, gdnf and Amh;
2) Identifying expression of a testicular support cell Sertoli cell specific protein in the testicular support cell (CiSCs), wherein the testicular support cell Sertoli cell specific protein is selected from the group consisting of SOX9, WT1, ZO-1, and CX43;
3) Identifying expression of the Sertoli cell surface receptor protein FSHR and/or AR in said testicular support cells (CiSCs);
4) Identifying secretion of trophic factors GDNF and INHIBIN in said testicular support cells (CiSCs); and/or
5) Identifying a testis support cell-related biological function of the testis support cell (CiSCs), wherein the testis support cell-related biological function is selected from the group consisting of cell migration ability, cell aggregation ability, ability to recruit endothelial cells, ability to induce cells to form tubular structures, ability to construct a seminiferous tubule structure in vitro, ability to provide a growth microenvironment for seminoma cells, ability to phagocytose germ cells, immunosuppressive ability (inhibition of T cell proliferation and IL-2 production), ability to produce tubular structures in subcutaneous grafts, and role in cell testis transplantation.
7. Testis-like support cells (CiSCs) obtainable by the method according to any of the above 1-6.
8. Use of testicular support cells (CiSCs) according to 7 above in research of reproductive developmental mechanisms, reproductive toxicology assessment, drug development and/or in vivo transplantation therapy.
9. A kit for reprogramming mammalian fibroblasts to testicular-like support cells (CiSCs), comprising:
1) A BET bromodomain inhibitor, preferably I-BET151;
2) Glycogen synthase kinase (GSK-3) inhibitors, preferably Riluzole (Riluzole); and
3) Retinoic Acid (RA).
Use of a bet bromodomain inhibitor, a glycogen synthase kinase (GSK-3) inhibitor, and Retinoic Acid (RA) in the preparation of a kit or pharmaceutical composition for reprogramming mammalian fibroblasts to testicular-like support cells (CiSCs).
More specifically, the invention utilizes three small molecule compounds I-BET151, riluzole, and RA (IRR) to induce fibroblast reprogramming into testis support cells, comprising the following steps: the primary fibroblast obtained by separation is passaged into a 6-hole plate, and the number of cells in each hole is 1-3 multiplied by 10 5 Culturing in DMEM medium containing Fetal Bovine Serum (FBS) at volume concentration of 10%, and treating fibroblast with 10 μm I-BET151 for 4 days after cell growth to logarithmic phase, wherein cell morphology is changed from original long spindle shape to flat shape, and cell gap is reduced to show epithelial cell characteristics; then the medium was replaced with a Sertoli cell induction medium containing 3. Mu.M Retinoic Acid (RA) and 10. Mu.M Riluzole, after which the medium was replaced every 3 days for induction for 25 days to obtain testis-like support cells having testis support cell characteristics.
The primary fibroblasts may be extracted from animals and selected from embryonic fibroblasts, visceral fibroblasts or skin fibroblasts. Methods for isolating fibroblasts are routine in the art.
The Sertoli cell induction culture is preferably DMEM medium containing 10% FBS,50ng/mL insulin-like growth factor, 50ng/mL epidermal growth factor, 50ng/mL fibroblast growth factor 9,0.1 μg/mL follicle stimulating hormone and 0.5 μg/mL prostaglandin D2.
The invention detects testis Support Cell (SCs) marker genes and protein expression levels of testis Support Cell (SCs) obtained by reprogramming for 25 days, and evaluates biological functions of the CiSCs, thereby comprehensively identifying the development state of the CiSCs. The results indicate that CiSCs are not only capable of expressing supporting cell marker genes and proteins, but also have similar biological functions as Sertoli cells, including the ability to migrate, aggregate, spontaneously form a tubular network structure, maintain seminoma survival in vitro, phagocytize apoptotic spermatogenic cells, and immunosuppression. CiSCs are transplanted into the testes and subcutaneously of mice and can be involved in the formation of seminiferous tubules of the testes and in the reconstruction of ectopic tubular structures. The above results indicate that CiSCs exhibit primary testicular support cell status.
The reprogramming testis-like support cell obtained by the invention can be used as a cell model for researching reproductive development mechanism, evaluating reproductive toxicology and researching and developing medicines related to Sertoli cells, and can also be used as a seed cell for in vivo transplantation related to Sertoli.
The invention demonstrates that the fibroblast chemistry reprogramming is carried out to construct a testicle supporting cell model and the functions thereof, and has the following advantages:
1) The invention utilizes three small molecular compounds to induce the fibroblast to be reprogrammed into the testis support cell, avoids the exogenous DNA caused by gene operation from interfering with genome, and has simple operation and high induction efficiency.
2) The reprogramming method provided by the invention is direct reprogramming, does not pass through a multipotent stem cell stage, and avoids the teratoma risk and ethical problems caused by stem cells.
3) The reprogrammed testis-like supporting cell obtained by the invention is a functional testis supporting cell, has an expression profile and biological function similar to those of an endogenous supporting cell, can be used for drug screening and reproductive toxicity evaluation by taking the testis supporting cell as a target cell, and has a wide application prospect in clinical transplantation treatment related to Sertoli cells.
Drawings
FIG. 1, schematic representation of small molecule compound-induced reprogramming of fibroblasts into testis-supporting cells, and morphology of testis-like supporting cells after reprogramming (MEFs-mouse fibroblasts; ciSCs-testis-like supporting cells; SCs-testis supporting cells).
FIG. 2, ciSCs express Sertoli cell-specific genes and proteins. (A) q RT-PCR detects the expression of Sertoli cell-associated mRNA in CiSCs, MEFs and SCs, including Wt1, krt18, fshr, ar, gdnf and Amh. (B) Immunofluorescent staining examined the expression of Sertoli cell-specific proteins (SOX 9, WT1, ZO-1 and CX 43) in CiSCs, MEFs and SCs. Scale bar = 20 μm. (C) Western Blot analysis of the expression of Sertoli cell surface receptor proteins (FSHR and AR) in CiSCs. (D) ELISA analysis showed that CiSCs can secrete trophic factors including GDNF and INHIBIN.
FIG. 3, migration capability of CiSCs. (A-B) CiSCs healed scratches rapidly in the scratch test and had higher mobility within 12 hours and 24 hours. (C-D) Transwell migration experiments demonstrated that CiSCs had a greater number of transmembrane cells within 24 hours. Membrane pore size = 8 μm.
Fig. 4, ciSCs, have the ability to aggregate, attract endothelial cell migration and spontaneously form a tubular network. (A) Soft agar colony formation experiments verify the aggregation capacity of CiSCs. (B-C) mobility determination of HUVECs incubated in conditioned Medium collected from MEFs, ciSCs and SCs. (D) Ability of MEFs, ciSCs and SCs to form a network after 4 hours of incubation on Matrigel. (E-G) CiSCs spontaneously form 3D tubule-like structures in vitro. (E) in vitro tubular structure construction experiment figures. (F) Bright field and H & E staining from MEFs, ciSCs and SCs reconstructed tissue. Scale bar = 10 μm. (G) immunofluorescent staining of reconstructed tissue sections. ZO-1, green; DAPI, blue. Scale bar = 10 μm.
FIG. 5, ciSCs, are capable of supporting germ cell survival in vitro, phagocytic apoptotic germ cells, and inhibiting T cell proliferation and IL-2 production. (A-B) MIFARE and MIFARE cell number statistics after 6 days co-culture with primary isolated mouse MIFARE cells with MEFs, ciSCs and SCs as feeder layers. Scale bar, 100 microns. (C) Representative images of lipid droplets in MEFs, ciSCs and SCs 3 days after co-culture with apoptotic sperm producing cells. The ORO staining was used to assess the ability to phagocytose apoptotic cells. Scale bar = 20 μm. (D-E) analysis of proliferation levels of primary mouse lymphocytes treated with MEFs, ciSCs and SCs conditioned Medium, respectively, and ELISA was used to detect IL-2 production.
FIG. 6, ciSCs in vivo transplantation. (A) CiSCs xenografts produce tubule-like structures. Cell clusters of MEFs, ciSCs and SCs were transplanted subcutaneously into NOD/SCID mice, respectively. Three weeks after injection, the grafts were removed, fixed and H & E stained. (B) After testicular transplantation, ciSCs form a seminiferous tubule in cooperation with endogenous support cells. Fluorescently labeled cells (CFDA-SE) were injected into testes of 2 week old mice. When the mice were adult, testes were removed and frozen sections were made. Fluorescent cells were observed for distribution by fluorescence microscopy after DAPI staining. Scale bar = 20 μm.
FIG. 7, ciSCs expression profiling. (A) Venn diagram analysis of the subset of genes shared by CiSCs and SCs. (B) Statistics of the number of differentially expressed genes in MEFs, ciSCs and SCs (DEGs, FPKM values, fold change)>2). (C) Pearson correlation metric was used to compare the similarity of transcriptional profiles of MEFs, ciSCs and SCs. (D) RNA-seq data analysis of heat maps of differentially expressed genes in MEFs, ciSCs and SCs. Color scale represents log of gene expression 2 A scale. Red and blue represent up-and down-regulated genes, respectively. (E) The scatter plot shows that in CiSCs, most of the support cell markers are activated and the fibroblast markers are silenced. Normalization of FPKM values, i.e., log, of sample differential genes 2 (FPKM+1). (F) GO terms of the first 10 GO functional analyses in CISC compared to MEF. (G) qRT-PCR analysis of expression of Sertoli-related genes and fibroblast-related genes in MEFs, ciSCs and SCs.
FIG. 8, immunofluorescence assay to evaluate the response of CiSCs to reproductive toxicants. CiSC and SCs were treated with known reproductive poisons (IL-1 a, busulfan, cadmium and Olaquindox) or controls (0.1% dmso), respectively, for 48 hours. Immunofluorescent staining detects disruption of the reticulation and supporting cell-specific protein ZO-1 to assess reproductive toxicity. DAPI, blue; ZO-1, green. Scale bar = 100 μm.
Detailed Description
Unless otherwise indicated, terms used herein have the ordinary technical meaning as understood by those skilled in the art.
The invention is further illustrated in the following examples by reference to the accompanying drawings. These examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The reagents used in the following are all commercially available products unless otherwise indicated.
Example 1: small molecule compounds induce reprogramming of mouse fibroblasts into Sertoli cells
(1) Preparation of mouse fibroblasts (MEFs): selecting 13.5 day mouse embryo (Kunming mouse, purchased from Guangdong province animal center), removing head, tail, limbs and viscera, cutting the rest tissue, digesting with 0.25% trypsin for 10min, adding double volume of DMEM medium containing 10% FBS to stop digestion, repeatedly blowing to give single cell suspension, inoculating cell suspension into 10cm culture dish at 37deg.C, and 5% CO 2 Culturing for 4 hours, then replacing fresh culture medium for continuous culture, and carrying out passage once after 2 days to finally obtain the fibroblast (MEFs).
(2) Small molecule compounds induce reprogramming of mouse fibroblasts into testis support cells: mouse fibroblasts (MEFs) were passaged into 6-well plates with a cell number of 3X 10 per well 5 In this case, the cells were grown after 24 hours in DMEM medium containing 10% FBS by volume and grown to the logarithmic phase using 10. Mu.M of I-BET151 (C 23 H 21 N 5 O 3 GSK1210151A, available from Selleck company) treated fibroblasts for 4 days, then the medium was replaced with a Sertoli cell induction medium (containing 10% FBS,50ng/mL insulin-like growth factor, 50ng/mL epidermal growth factor, 50ng/mL fibroblast growth factor 9,0.1. Mu.g/mL follicle stimulating hormone and 0.5. Mu.g/mL prostaglandin D2 medium) containing 3. Mu.M Retinoic Acid (RA) (available from Selleck company) and 10. Mu.M Riluzole (Riluzole) (available from Selleck company), after which testicular support cells (CiSCs) having testicular support cell characteristics were obtained by replacing the medium once every 3 days for induction for 25 days.
The method for inducing mouse fibroblast to reprogram into testis support cell by small molecule compound is shown in figure 1, and the induced testis support cells are arranged in a rope shape with clear boundary, which is similar to the form of primary support cell SCs (separation method is described below).
Isolation of primary support cell SCs in mice: male Kunming mice were sacrificed at about 4 weeks of birth, and sterilized by soaking in 75% alcohol for 10min. The testis (without damaging the testis white membrane) was removed and placed in sterile PBS (containing 1% diab) pre-chilled in advance, and washed four times with PBS repeatedly; then, the testis membrane was torn off using replaced sterile forceps, the seminiferous tubules were removed, placed in a new bacterial dish containing sterile PBS, and repeatedly pulled with forceps to loosen the seminiferous tubules. The seminiferous tubules were then placed in a 50mL centrifuge bowl, 0.5mg/mL collagenase IV (purchased from Sigma) was added, digested with shaking at 37℃for 15min, and mixed well. After the tissue subsides naturally, the supernatant is discarded to remove the mesenchymal cells. Then, 5mL of collagenase IV (purchased from Sigma) at a concentration of 0.5mg/mL was added, and the mixture was allowed to stand and digest for 20 minutes, and the mixture was passed through a 100 μm filter membrane and washed with sterile PBS multiple times to remove myoid cells. Finally, the scraped tissue mass was placed in a 15mL centrifuge tube, 1mg/mL hyaluronidase 4mL was added, and the seminiferous tubules were completely dispersed by repeated blowing using a glass pipette. Digestion is carried out at 37℃for 5min and centrifugation is carried out at 1000rpm for 5min. The supernatant was discarded and resuspended in 5mL of DMEM medium containing 10% serum and diabody. The cell suspension was transferred to a 90mm cell culture dish, continuously cultured in an incubator at 37℃for 48 hours, the medium was discarded, and 0.1M Tris-HCl hypotonic solution was added for 2 minutes to remove sperm-like cells. Fresh DMEM complete medium is replaced, the culture is continued for 48 hours at 37 ℃, and the culture medium can be used for experiments after the cells grow fully.
Example 2: qRT-PCR detection of testicular support cell gene expression induced by small molecule compound
Mouse fibroblasts were induced to reprogram into testis-like support cells according to the procedure of example 1, ciSCs cells were collected, total RNA was extracted from the cells using HiPure Total RNA Mini Kit (R4111-02, magen), and 1 μg of RNA was reverse transcribed into cDNA using RT Master Mix (RR 036A, taKaRa), then cDNA samples were mixed with 2× ChamQ SYBR qPCR Green Master Mix (Q311-02, vazyme) and real-time quantitative PCR analysis was performed using CFX Connect real-time PCR detection system (Bio-rad). PCR was performed for 40 cycles using the primers described in Table 1. The relative expression levels were normalized to those of the internal control (GAPDH) and confirmed in at least 3 separate assays.
As shown in FIG. 2A, ciSCs are capable of expressing testis support cell-associated genes, including Wt1, krt18, fshr, ar, gdnf and Amh.
Example 3 Induction of expression of testis-like supporting cell proteins by Small molecules
Immunofluorescence detection of the seltoli cell-characteristic protein: testis-like support cells were obtained as in example 1, MEFs, ciSCs and SCs were spread on cell slide plates, respectively, and after incubation for 24 hours, the cell slide plates were fixed with 4% formaldehyde (PFA) for 10min, immersed in PBS buffer for 5min X3 times, 0.2% Triton X-100 was allowed to permeate for 30min, blocking buffer (P0102, beyotide) was incubated at room temperature for 1 hour, primary antibody (see Table 2) was added, incubated overnight at 4℃in a wet box, then washed with PBS buffer for 7min X3 times, and secondary antibody (see Table 2) (Goat Anti-Rabbit IgG H)&L(Alexa488 Available from Abcam corporation), incubated for 1 hour at room temperature, and observed by fluorescence microscopy.
As shown in FIG. 2B, ciSCs were able to express testis-supporting cell characteristic proteins, including WT1 (NP-659032.3), sox9 (NP-035578.3), ZO-1 ((NP-033412.2) and CX43 (NP-034418.1).
Western blot detection of Sertoli cell characteristic protein: ciSCs were obtained as in example 1, cellular proteins were extracted as usual, run on 12% sds-PAGE, and then transfer to a TBST buffer of 5% skim milk powder after transfer, blocking for 1 hour at room temperature, washing 5min×3 times with TBST buffer, adding primary antibody (from Abcam corporation), incubating overnight at 4 ℃, then washing 5min×3 times with TBST buffer, adding secondary antibody (from Bioworld corporation), incubating for 1 hour at room temperature, washing 5min×3 times with TBST buffer, and photographing with developer.
As shown in FIG. 2C, ciSCs were able to express testicular support cell surface receptor proteins, including FSHR (NP 038551.3) and AR (NP 038504.1).
Elisa assay Sertoli cell-associated secreted proteins: ciSCs were obtained as in example 1, and the supernatant of the cell culture was collected and subjected to lyophilization, followed by detection with GDNF test kit (from Jiangsu Nanjing institute of biological engineering, cat# H071) and INHB test kit (from Jiangsu Nanjing institute of biological engineering, cat# H320-2).
As a result, as shown in FIG. 2D, ciSCs are able to secrete trophic factors GDNF (NP 001288286.1) and INHIBIN (NP 032407.1).
Example 4 identification of testis support cell-related biological Functions of small molecule-induced testis-like support cells
Testis-like support cells (CiSCs) were obtained as in example 1 for identification of testis support cell-related biological functions.
1) Scratch experiments detect the migration ability of cells: the day before the experiment, a marker was used to draw parallel lines on the back of the 6-well plate to ensure that three parallel lines were run through each well. After the experimental cells were digested into single cell suspensions, the foregoing CiSCs, MEFs and SCs were mixed at 1X 10 6 The individual cell/well concentrations were cultured overnight in 6-well plates. The next day, cells were scraped with 200 μl of parallel lines drawn perpendicular to the bottom of the plate, washed twice with PBS to remove cell debris, and then placed in serum-free medium. Photography was performed at 0 and 24 hours after incubation, and the average distance of scratches at each time point was measured.
As a result, as shown in FIG. 3 at A, B, at 12h post-scratch, the scratch width of CiSCs was significantly narrowed relative to MEFs and the healing rate was relatively close to that of SCs, with MEFs scratch width did not change much as the experiment was extended to 24h, while the scratches of CiSCs and SCs were substantially healed.
2) Transwell migration experiments further verify the migration ability of CiSCs: taking logarithmic growth cells, discarding the culture medium, washing twice with PBS, adding a serum-free culture medium, and starving for culturing for 12-24 hours; after digestion, the cells were washed 1 to 2 times with PBS and resuspended in 1% BSA-containing medium at a cell concentration of 1 to 5X 10 5 100. Mu.L (2X 10) of the sample was taken out per mL 4 ) The cell suspension was added to the upper chamber of the Transwell chamber, and 500. Mu.L of a cell culture medium containing 10% FBS was added as the lower chamber to the 24-well plate, and cultured in an incubator at 37℃for 48 hours; taking out the cell, sucking off culture medium in the upper and lower chambers, wiping off cells inside the upper chamber with cotton bud, adding 500 μl of 4% paraformaldehyde into the lower chamber, fixing cells at room temperature for 15min, discarding the fixing solution, soaking in PBS, cleaning for 5min, adding into the lower chamberAdding 500 mu L of DAPI staining solution, staining for 10min at room temperature in dark, soaking and cleaning with ultrapure water for 5min, repeating for 3 times, dripping anti-fluorescence quenching agent on the glass slide, placing the cell on the glass slide, shooting under a fluorescence microscope, and calculating the cell number.
As a result, as shown in FIG. 3 at C, D, the number of migrating cells of CiSCs and SCs was significantly higher than MEFs, and the number of migrating cells of CiSCs was 3 times that of MEFs with statistically significant differences when three groups of transmembrane cells were counted.
3) Soft agar clone formation verifies cell aggregation ability: mixing sterile 5% agarose at 40deg.C with DMEM medium preheated at 37deg.C at a ratio of 1:9, adding the mixture into 12-well plate with a volume of 1mL per well, standing at room temperature for solidification, mixing single cell suspension with 0.5% agarose preheated at 40deg.C at equal volume, adding cell-agarose mixture into 12-well plate pre-coated with agar, and collecting cell number of 1×10 per well 4 After standing at room temperature until complete solidification, plates were placed in an incubator for cultivation for 24 hours, 200. Mu.L of medium was added to each well and replaced every other day, and cultivation was continued until colony appeared, and the colonies were stained with an alkaline phosphatase assay kit (available from Shanghai Biyun biotechnology Co., ltd., cat# product No. P0321S) and photographed to calculate the number of clones.
As shown in FIG. 4A, ciSCs and primary support cells formed spheroid aggregates, but MEFs failed to form cell aggregates.
4) Transwell assays induce the ability of cells to recruit endothelial cells: will be 2X 10 5 CiSCs, MEFs and SCs were grown in 1 well of a 6-well plate, and after 24 hours of culture, the original medium was replaced with serum-free DMEM, and the cells were further cultured for 48 hours, and then the supernatants were collected as conditioned medium, respectively. Will be 1X 10 4 Individual umbilical endothelial cells (HUVEC) (laboratory preservation) were seeded on a microporous membrane (8 mm) Boyden chamber containing serum-free DMEM in the upper part and 50% conditioned medium (600 μl total) in the lower part and incubated for 12 hours. Subsequently, cells in the inner chamber were wiped off while fixing the cells migrating through the membrane with 4% formaldehyde (PFA) at Room Temperature (RT) for 15 minutes and stained with DAPI at room temperature for 10 minutes. Migration measurement by computing DAPI blue signal for each imageAnd (3) cells.
As a result, as shown in FIG. 4 at B, C, after 24 hours of co-culture, the number of HUVECs attracted by SCs and CiSCs and passing through the underside of the membrane was 2-fold higher than MEFs.
5) Demonstration of the ability of induced cells to form tubular structures: matrigel (available from BD Co., U.S. under the trade name 356234) was mixed with DMEM medium containing 5% FBS in equal volume, spread on a pre-chilled 24-well plate, and coagulated in a 37℃incubator for 1 hr to obtain 1X 10 gel 6 The individual cells were seeded onto the coagulated matrigel, allowed to stand in an incubator for 4 hours, and the network structure was observed with a microscope and photographed.
As a result, both CiSCs and SCs can form a network on matrigel consistent with the onset of tubular formation, as shown in FIG. 4D.
6) 3D culture in vitro construction of a similar aspergillosis seminiferous tube structure: the cell suspensions were dropped onto the lid of the petri dish and incubated upside down at 34℃for 2 days, wherein each drop of suspension contained 1X 10 6 Individual cells/20. Mu.L, while mixing 20ml of sterile 0.75% agarose solution pre-heated at 40℃with an equal volume of 2 XDMEM medium, pouring into a petri dish and cooling to room temperature, and slicing the solidified mixture into 1cm 3 Is transferred to wells of a 24-well plate, the agarose blocks are immersed in DMEM complete medium, placed overnight in a 37 ℃ incubator to exchange nutrients in the blocks, then the cell clusters cultured in the suspension droplets for 48 hours are transferred to the surface of the agarose blocks, fresh medium is replaced to ensure that the liquid level is parallel to the upper surface of the agarose blocks, further cultured in a 34 ℃ incubator, the medium is updated every other day during which time, the cell clusters are collected every 14 days, 4% paraformaldehyde is fixed for 10 minutes at room temperature, then frozen sections of 10 μm thickness are made, HE staining observes the internal morphology of the cell clusters, immunofluorescent staining (agat Anti-rabit IgG H&L(Alexa488, available from Abcam corporation) detects the expression of ZO-1 (np_ 033412.2) in the cell cluster.
As a result, as shown in FIG. 4 at E, F, G, ciSCs and SCs show a pronounced tendency for cell mass growth relative to the radioactive, loose structure of MEFs and develop a relatively compact sphere structure. After HE staining, it was observed that the mass formed by CiSCs appeared to have a relatively smooth and dense exogenous profile, the internal cell shape had a chemotactic cord structure, and a distinct network structure was observed by enlarging the cord, which was quite similar to the state of SCs. Upon immunofluorescent staining, cell regularity interconnections can be observed in the cell mass composed of CiSCs, and ZO-1 protein forms a basal membrane around the cell chain.
7) CiSCs co-culture with spermatogonia: ciSCs, MEFs and SCs were treated with medium containing 10. Mu.g/ml mitomycin for 3 hours and the treated cells were used as feeder cells at 5X 10 5 The number of individual cells/well was inoculated into 6-well plates coated with 1% gelatin and cultured for 24 hours. The sperm cells separated for 24 hours were then isolated according to 1X 10 6 The ratio of/well was seeded into 6-well plates with feeder cells laid out, and the culture was continued in an incubator during which the medium was refreshed every 2 days, and after 6 days of culture, the growth of the sperm cells was observed and photographed in a bright field.
8) Separation of spermatogonia: in example 1, primary supporting cells were isolated and cultured, after 48 hours of primary culture, spermatogenic cells suspended in the culture solution were collected, transferred to a new cell culture dish, and cultured at 37℃for 2 days to obtain spermatogenic cells;
as a result, as shown in FIG. 5 at A, B, after 6 days of co-culture with sperm cells, a greater number of spermatogenic cell clusters were observed in CiSCs than in MEFs, and there was no significant difference in the number of cell clusters between CiSCs and endogenous supporting cells. The results demonstrate that CiSCs can provide a better microenvironment for the survival of sperm cells than MEFs.
8) Phagocytic capacity identification of CiSCs: the primary support cells were isolated 24 hours after, the spermatogenic cells suspended in the medium were collected and cultured for 3 days in the absence of feeder layers to induce spontaneous apoptosis, and the apoptotic cells were washed with 1 XPBS and resuspended in DMEM for phagocytosis assay. CiSCs, MEFs and SCs were combined at 5X 10 4 Individual cells/well were seeded on glass slides in 24 well plates and after 24 hours of incubation, apoptotic sperm cells were grown at 1X 10 6 Adding individual cells/wells into well plate, and co-culturing spermatogonial cells with CiSCsAfter 3 days of culture, apoptotic cells were removed and cells plated on glass coverslips were stained with ORO.
As a result, as shown in FIG. 5C, a large number of red-colored lipid droplets appeared in CiSCs and SCs after oil red O staining, while few lipid droplets were observed in MEFs, indicating that the phagocytic activity of CiSCs was significantly higher than that of MEFs.
9) Identification of immunosuppressive Capacity of CiSCs: the immunosuppressive ability of cells was assessed by inhibiting lymphocyte proliferation and interleukin-2 production. Will be 2X 10 5 CiSCs, MEFs and SCs were planted in one well of a 6-well plate, cultured in an incubator at 37℃for 24 hours, replaced with serum-free DMEM, and after further culturing for 48 hours, the supernatants were collected as conditioned medium, respectively. For lymphocyte proliferation assays, conditioned medium was diluted to 20%, 40%, 60% and 80% with DMEM medium, and 10% pbs was added, respectively. Lymphocytes were isolated from spleens of male Kunming mice (purchased from the animal center in Guangdong province) of 6 to 8 weeks old, mixed with a conditioned medium of prescribed concentration, inoculated into 96-well plates at 2X 10 cells/well, placed at 37℃for further 48 hours, and proliferation of lymphocytes was measured by CCK-8 kit (purchased from MCE Co., ltd., cat# HY-K030). The production of interleukin-2 in lymphocytes was analyzed by mixing lymphocytes with respective 40% medium under different cell conditions at a concentration of 1X 10 6 Individual cells/wells were seeded in 6-well plates and cultured for 2 days, lymphocytes were collected and lysed with RIPA cell lysate, and the concentration of IL-2 in the lymphocyte lysate was determined by ELISA kit (ex institute of bioengineering, build H003, from singeing, south of the Jiangsu).
The results are shown in figure 5 as D, E; compared to MEFs conditioned medium, the medium from CiSCs or SCs was able to significantly inhibit lymphocyte proliferation and the IL-2 levels in lymphocytes were low, suggesting that CiSCs could inhibit T cell proliferation and IL-2 production in vitro.
Example 5: in vivo transplantation of small molecule-induced testicular support cells
Testicular support cells (CiSCs) were obtained as in example 1 for in vivo transplantation experiments.
(1) The creation of tubular structures within subcutaneous implants: MEFs, ciSCs and SCs clusters were subcutaneously injected into the backs of NOD/SCID mice (purchased from animal centers, guangdong province) and kept for three weeks, subcutaneous tissue blocks were removed, 4% paraformaldehyde was fixed, frozen sections were prepared, and tissue structures were observed by HE staining.
As a result, as shown in FIG. 6A, the formation of tubular structures was observed in grafts of CiSCs and SCs 3 weeks after transplantation, as compared with MEFs.
(2) Cell testis transplantation: cells were transplanted into testes of new 10 day old Kunming mice (purchased from animal centers in Guangdong province) to identify whether the cells were involved in the construction of testis seminiferous tubules. Using CFSE cell proliferation assay and tracking kit (Kaiyi, cat# KGA 318) fluorescence labeled MEFs, ciSCs and SCs, after anesthetizing the mammalian mice, a wound of about 1cm was excised from the lower abdomen of the mammalian mice, and the left testis was gently pulled out with forceps, under a stereo microscope, 1X 10 was performed with glass microneedles 6 Fluorescently labeled cells were injected into the testes, the testes injected with labeled cells were placed back into the abdominal cavity, then the incision was sutured and sterilized with iodophor, and when recipient mice were awake, they were placed back in the cage and housed with the master mice. One month after implantation, the recipient mice developed to maturity, testes were obtained and frozen into sections, and after DAPI staining, the distribution of green fluorescent cells in the testes was observed and photographed.
As a result, as shown in FIG. 6B, two weeks after implantation, ciSCs integrated into and colonized the basal portion of the recipient's seminiferous tubules. In contrast, MEFs are distributed between the seminiferous tubules.
EXAMPLE 6 expression profiling
Testis-like supporting cells (CiSCs) were obtained as in example 1, cells were collected, total RNA was extracted by Trizol method, and the quality and concentration of the samples were checked using Nano Drop ND-1000RNA (Invitrogen, cat# 15596018), requiring A260/A280 to be 1.8 or more and A260/A230 to be 1.5 or more in quality control, ensuring that the samples were free from contamination, and checking RIN value of the samples to determine RNA integrity, wherein RIN to be 7.0 indicates that the sample integrity was good. Determination of RNA DNA from Total RNA was digested with DNase I and the digested product was purified with magnetic beads. For eukaryotes, mRNA was enriched with Oligo (dT) beads and for prokaryotes, rRNA was removed with a kit to obtain enriched mRNA. Uniformly mixing mRNA with a proper amount of breaking reagent, breaking under the action of high temperature, and carrying out reverse transcription by using segmented mRNA as a template and using a six-base random primer to synthesize double-stranded cDNA; end repair, polyA addition, sequencing linker addition: the end repair complex enzyme performs end repair on the double-stranded cDNA. The repair product was purified with magnetic beads, end-added with A under the action of polymerase and ligated with sequencing adaptors under the action of ligase. The ligation products were amplified by configuring a PCR reaction system and purified using magnetic beads, and quality and yield were detected by Agilent 2100 Bioanalyzer and real-time fluorescent quantitative PCR, and sequenced on-machine until the library construction was completed.
The results are shown in FIG. 7: the Venn diagram analysis showed that 16982 genes were shared between CiSCs and SCs (FIG. 7, A). The results of differential gene number analysis showed that a total of 4552 genes were differentially expressed between CiSCs and MEFs, including 2337 genes up-regulated and 2175 genes down-regulated, while 4818 genes were differentially expressed between SCs and MEFs, including 2618 up-regulated and 2200 down-regulated genes (fig. 7, b). Person correlation and hierarchical clustering analysis showed that CiSCs aggregated with endogenous Sertoli cells (fig. 7, c). Compared with MEFs, 466 genes in CiSCs are up-regulated, 748 genes are down-regulated, and the expression level is changed by more than 2 times. The global gene expression pattern of CiSCs is more similar to that of adult support cells (7,D). The scatter plot shows that genes up-regulated in CiSCs are significantly enriched in SCs, while down-regulated genes are enriched in MEFs (fig. 7, e). GO functional analysis showed that differential genes are associated with supporting cellular function such as cell surface, extracellular space, extracellular matrix and cell attachment (figure 7,F). Consistent with the expression profile, qRT-PCR further showed that the Sertoli cell-associated gene was strongly activated in CiSCs, while the amount of fibroblast marker gene expression was significantly reduced, and the global gene expression pattern of CiSCs was similar to that of mature support cells, unlike that of primary fibroblasts (fig. 7,G).
Example 7 application of small molecule induced testis-like support cells in evaluation of reproduction toxicity
According to realityEXAMPLE 1 procedure Induction of reprogramming of mouse fibroblasts into testis-like support cells at 1X 10 5 The individual cells/wells were seeded on glass slides in 24 well plates, placed in a 37℃incubator for 24 hours, the original medium was replaced with medium containing different reproductive toxicants, and the culture was continued for 48 hours, the supernatant was discarded, and the cell response to reproductive toxic substances was judged by immunofluorescence detection of the expression of Zona Occludes 1 protein (ZO-1) in the cells. The final solvent concentration was consistent under all conditions and all compounds tested were reported to have reproductive toxicity. Details of the test compounds are listed in table 3.
As shown in FIG. 8, ciSCs showed a broken ZO-1 distribution pattern after treatment with a reproduction toxic substance, and ZO-1 expression was significantly disrupted with no significant difference in the degree of disruption of the primary testicular supportive cytotoxicant. These findings indicate that induced class of support cells (CiSCs) are sensitive to reproductive poisons and can be an alternative model for evaluating the deleterious effects of substances on male fertility.
It will be appreciated by persons skilled in the art that although the invention has been specifically described with reference to the above embodiments, the invention is not limited to these specific embodiments. Based on the methods and technical solutions taught by the present invention, those skilled in the art can make appropriate modifications or improvements without departing from the spirit of the present invention, and the equivalent embodiments thus obtained are within the scope of the present invention.
TABLE 1 quantitative PCR primers
TABLE 2 antibody information
TABLE 3 information on reproduction toxic Compounds
Industrial applicability
The invention demonstrates the construction of the testicle support cell model prepared by the reprogramming method and the application thereof, and proves that the constructed cell model can be used as a substitute model for researching the physiological function of the testicle support cell, so as to be applied to mechanism research, drug screening and toxicology evaluation which take the testicle support cell as a target cell, and the constructed cell can also be used in the related transplantation treatment which takes the testicle support cell as a seed cell. The method is easy to operate and high in efficiency, does not introduce exogenous genes, is hopeful to be developed into a widely applied method for obtaining the non-testis-source support cells, not only provides a cell model and a theoretical basis for basic research and drug development of reproductive system diseases, but also brings wide prospects for application of testis support cells in the field of regenerative medicine.
Claims (2)
1. A method of reprogramming/inducing mammalian fibroblasts into testicular-like support cells (CiSCs), the method comprising the steps of:
(1) Treating isolated mammalian fibroblasts with a BET bromodomain inhibitor to change the morphology of the cells from original long fusions to flattened shapes; and
(2) Further treating the cells from step (1) with a glycogen synthase kinase (GSK-3) inhibitor and Retinoic Acid (RA) to thereby obtain said testicular support cells (CiSCs), wherein said BET bromodomain inhibitor is I-BET151 and said glycogen synthase kinase (GSK-3) inhibitor is Riluzole,
wherein the mammalian fibroblast is a mouse embryonic fibroblast,
the method comprises the following specific steps: the primary fibroblast obtained by separation is passaged into a 6-hole plate, and the number of cells in each hole is 1-3 multiplied by 10 5 Culturing in DMEM medium containing Fetal Bovine Serum (FBS) at volume concentration of 10%, and treating fibroblast with 10 μm I-BET151 for 4 days after cell growth to logarithmic phase, wherein cell morphology is changed from original long spindle shape to flat shape, and cell gap is reduced to show epithelial cell characteristics; then the medium was replaced with a Sertoli cell induction medium containing 3. Mu.M Retinoic Acid (RA) and 10. Mu.M Riluzole, after which the medium was replaced every 3 days for induction for 25 days to obtain testis-like support cells having testis support cell characteristics.
2. The method of claim 1, the method further comprising:
1) Identifying expression of a testis support cell-related gene in said testis-like support cells (CiSCs), wherein said testis support cell-related gene is selected from the group consisting of Wt1, krt18, fshr, ar, gdnf and Amh;
2) Identifying expression of a testicular support cell Sertoli cell specific protein in the testicular support cell (CiSCs), wherein the testicular support cell Sertoli cell specific protein is selected from the group consisting of SOX9, WT1, ZO-1, and CX43;
3) Identifying expression of the Sertoli cell surface receptor protein FSHR and/or AR in said testicular support cells (CiSCs);
4) Identifying secretion of trophic factors GDNF and INHIBIN in said testicular support cells (CiSCs); and/or
5) Identifying a testis support cell-related biological function of the testis support cell (CiSCs), wherein the testis support cell-related biological function is selected from the group consisting of cell migration ability, cell aggregation ability, ability to recruit endothelial cells, ability to induce cells to form tubular structures, ability to construct a seminiferous tubule structure in vitro, ability to provide a growth microenvironment for seminoma cells, ability to phagocytose germ cells, immunosuppression ability, ability to produce tubular structures in subcutaneous grafts, and role in cell testis transplantation.
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| JP6938154B2 (en) * | 2014-11-07 | 2021-09-22 | 国立大学法人大阪大学 | Differentiation-inducing cell population from which undifferentiated cells have been removed, its utilization and its production method |
| KR101986830B1 (en) * | 2017-09-07 | 2019-06-07 | 차의과학대학교 산학협력단 | Sertoli-like cells from stem cells, a method for preparing thereof, and uses thereof |
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| CN106636210A (en) * | 2016-10-09 | 2017-05-10 | 广州暨南大学医药生物技术研究开发中心 | Method for inducing transdifferentiation of fibroblast into similar testicular interstitial cells by combination of transcription factors |
| WO2020006675A1 (en) * | 2018-07-03 | 2020-01-09 | 清华大学 | Method for in vitro reprogramming of fibroblasts into sertoli cells and application thereof |
| CN109706114A (en) * | 2019-02-21 | 2019-05-03 | 吉林大学 | A kind of method that bull testis sertoli cell induces multi-potent stem cell |
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