STEM CELL-DERIVED SERTOLI-LIKE CELL, PREPARATION METHOD THEREFOR, AND USE THEREOF

Abstract

Provided are stem cells-induced Serotoli-like cells, methods of preparing the same, and uses thereof. Sertoli-like cells according to one embodiment can be differentiated from embryonic stem cells with excellent proliferative capacity, and thus, can be obtained in large quantities. Also, since the Sertoli-like cells secrete immunosuppressive substances and form immune privilege and induce anti-inflammatory functions, they can be used for development of the cell therapeutic agent.

Claims

1. A method of preparing Sertoli-like cells (SLC) from stem cells, the method comprising: inducing differentiation into SLC by culturing stem cells.

2. The method of claim 1, further comprising inducing differentiation into intermediate mesoderm (IM) by culturing stem cells.

3. The method of claim 2, wherein the inducing of differentiation into the IM comprises (a) culturing stem cells in a medium containing a GSK-3 inhibitor, and (b) additionally culturing after adding of bFGF and RA to the medium.

4. The method of claim 3, wherein in the process (a), the culturing period is one day to three days, and in the process (b), the culturing period is two days to five days.

5. The method of claim 3, wherein the medium in the process (a) further comprises L-glutamine, antibiotics, or a combination thereof.

6. The method of claim 1, wherein in the process of inducing differentiation into SLC, the culturing period is five days to seven days.

7. The method of claim 1, wherein the concentration of the GSK-3 inhibitor is from 1 M to 10 M, the concentration of bFGF is from 50 ng/ml to 200 ng/ml, the concentration of RA is from 0.5 M to 1.5 M

8. The method of claim 1, wherein the stem cells are embryonic stem cells (ESC), adult stem cells, or induced pluripotent stem cells (iPSC).

9. The method of claim 8, wherein the embryonic stem cells are embryonic stem cells of a mouse.

10. The method of claim 1, further comprising isolating or purifying induced SLC.

11. SLC prepared by using the method of claim 1.

12. The SLC of claim 11, wherein Wt1, Sox9, Sf1, Gata4, Fshr, Scf, or a combination thereof is expressed.

13. A method of treating or preventing an autoimmune disease comprising administering, to a subject in need thereof, an effective amount of SLC prepared by using the method of claim 1, or a cell population thereof, and a culture, lysate, or extract thereof.

14. The method of claim 13, wherein the autoimmune disease comprises at least one selected from rheumatoid arthritis, osteoarthritis, Behcet's disease, autoimmune cytopenia, autoimmune myocarditis, atopic dermatitis, asthma, primary cirrhosis, dermatomyositis, Goodfitz syndrome, autoimmune meningitis, Sjogren's syndrome, systemic lupus erythematosus, Addison's disease, alopecia areata, ankylosing spondylitis, autoimmune hepatitis, autoimmune mumps, Crohn's disease, diabetic disease, dystrophic epidermolysis bullosa, epididymitis, glomerulonephritis, Graves' disease, Guillain-Barr syndrome, Hashimoto's disease, hemolytic anemia, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, psoriasis, sarcoidosis, scleroderma, seronegative spondyloarthropathy, pernicious anemia, scleroderma, mitochondrial related syndrome, and inflammatory bowel disease.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0070] FIG. 1 illustrates a simplified diagram showing the method of specification from embryonic stem cells (ES) into intermediate mesoderm (IM) via mesendodem (ME) (CHIR: CHIR99021).

[0071] FIG. 2 shows a day-2 phase change image (D2) of mESC treated with CHIR, and day-4, 5, and 6 phase change images (D4, D5, D6) of mESC treated with bFGF and RA (CHIR: CHIR99021).

[0072] FIG. 3 shows the results of RT-PCR confirming mRNA expression levels of Oct4, Pax2, Osr1, Lhx2, and Wt1 on days 2, 4, 5, and 6 (D2, D4, D5, and D6) while mESC was induced into IM.

[0073] FIG. 4 shows the results of real time-PCR confirming mRNA expression levels of Oc4, Pax2, Lhx1, Wt1, and Osr1 on days 2, 4, 5, and 6 (D2, D4, D5, and D6) while mESC was induced into IM.

[0074] FIG. 5 shows the results of immunofluorescence staining for IM markers PAX2 and LHX1 in mESC-induced IM; Scale bar of 50 m.

[0075] FIG. 6 shows a schematic view illustrating a method of differentiating mESC-induced IM into SLC.

[0076] FIG. 7 shows phase change images of SLCs 3 days after differentiation from IM (D3 SLCs) and 7 days after differentiation from IM (D7 SLCs), and 5-day old mouse SCs, which are immature mouse SCs.

[0077] FIG. 8 shows images of SCs obtained from 5-day old mouse (5 d mouse SCs) and ESC-induced SLCs which were cultured in 10% FBS and a differentiation medium, the images confirming the morphology thereof.

[0078] FIG. 9 shows the results of RT-PCR confirming mRNA expression levels of Oct4, Wt1, Sox9, Sf1, Gata4, Fshr, and Scf in mESC, induced SLC, and SCs.

[0079] FIG. 10 is the graph showing the results of real time PCR confirming mRNA expression levels of Oct4, Wt1, Sox9, Sf1, Gata4, Fshr, and Scf in mESC, induced SLC, and SCs.

[0080] FIG. 11 shows immunofluorescence staining results for SC markers GATA4 and FSHR in SLC induced from IM; Scale bar of 50 m.

[0081] FIG. 12 shows an image showing the results of immunostaining of SLC with respect to FSHR and GATA4 before purification, isolation and after isolation; FSHR+: FSHR positive; FSHR: FSHR negative.

[0082] FIG. 13 shows a graph of the flow cytometry results before and after isolation.

[0083] FIG. 14 shows a graph showing the results of real time PCR confirming mRNA expression levels of Oct4, Wt1, Sox9, Gata4, and Fshr genes in FSHR-positive SLC and FSHR-negative SLC; mESCs: mouse embryonic stem cells; FSHR(+): FSHR-positive SLC; FSHR(): FSHR-negative SLC.

[0084] FIG. 15 shows images of the results of RT-PCR confirming mRNA expression levels of Oct4, Wt1, Sox9, Gata4, and Fshr genes in FSHR-positive SLC and FSHR-negative SLC; mESCs: mouse embryonic stem cells; FSHR(+): FSHR-positive SLC; FSHR(): FSHR-negative SLC; D.W: Distilled water (control).

[0085] FIG. 16 shows images of a testicle and seminiferous tubule of a mouse of which EGFP expression was confirmed by ultraviolet (UV) light after SLC transplantation; Scale bar of 5 cm.

[0086] FIG. 17 shows the results of immunofluorescence of testicles stained with anti-GATA4 (red) and anti-GFP (green) antibodies 1 and 7 days after SLC transplantation; Scale bar of 20 m.

[0087] FIG. 18 shows images showing phagocytosis activity of FSHR-positive SLC (FSHR (+)) and FSHR-negative SLC (FSHR ()). Blue: Hoechst-stained nucleus; Green: fluorescent microbeads; Red: induced SLC, derived from RFP-transplanted mESCs; FSHR (+): FSHR-positive SLC; FSHR (): FSHR-negative SLC; Scale bar of 40 m.

[0088] FIG. 19 shows a graph comparing phagocytosis activities of adult SC, 5-day old SC, FSHR-positive SLC, FSHR-negative SLC and MEF.

[0089] FIG. 20 shows a histogram measuring CD4.sup.+T cell proliferation via CFSE; hBM-MSCs: human bone marrow-induced mesenchymal stem cells; FSHR (+) SLCs: FSHR-positive SLC.

[0090] FIG. 21 shows a graph of the average value obtained by measuring CD4.sup.+ T cell proliferation through CFSE. hBM-MSC: human bone marrow-induced mesenchymal stem cells; FSHR (+) SLCs: FSHR-positive SLC.

[0091] FIG. 22 shows the results of RT-PCR confirming mRNA expression levels of TGF-1, Transferrin, IL-6, Fas-L and Clusterin in un-isolated SLC, FSHR-positive SLC, and adult testicles; FSHR(+) SLCs: FSHR-positive SLC; D.W; Distilled water (control).

[0092] FIG. 23 illustrates the graph showing the results of real time-PCR confirming mRNA expression levels of TGF-1, Transferrin, IL-6, Fas-L and Clusterin in un-isolated SLC, FSHR-positive SLC, and adult testicles; FSHR(+) SLCs: FSHR-positive SLC; D.W; Distilled water (control).

MODE OF DISCLOSURE

[0093] Hereinafter, the present disclosure will be described in more detail with reference to Examples. However, these examples are intended to illustrate the present disclosure and the scope of the present disclosure is not limited to these examples.

Example 1. Specification from Mouse Embryonic Stem Cells (ESC) to Intermediate Mesoderm (IM)

[0094] 1.1 Storage of Mouse ESC (mESCs)

[0095] mESC strain (karyotype: XY) was derived from C57BL/6 substrain mice and GFP-expressing transgenic mice [C57BL/6-Tg (CAG-EGFP), Japan SLC, Inc., Shuzuoka, Japan], and the mouse embryonic fibroblasts (MEFs; CF1 strain, Jackson Laboratory, Los GoTos, Calif.) were cultured as feeder cells. The cells were stored in a culture medium consisting of 80% (v/v) DMEM high glucose (HyClone, Logan, Utah) containing 20% (v/v) mESC, SR (Gibco-BRL, Frankin Lakes, N.J.), 1% (v/v) NEAA (Gibco-BRL), 0.1% (v/v) -mercaptoethanol (Gibco-BRL), 100 U/ml LIF (ESGRO, Chemicon, Temecula, Calif.), in an incubator at a temperature of an 37 C. in a 5% wet CO.sub.2. For passage, mESC was treated with 0.05% trypsin-EDTA (TE; HyClone) for 3 minutes, removed from the dish and divided into new MEF-seed dishes every 3 to 4 days. mESC culture medium was exchanged daily.

[0096] 1.2. Differentiation of mESC into IM and Confirmation of the Same

[0097] Undifferentiated mESCs stored as described above were seeded on an eltrex (Gibco-BRL)-coated plate at a density of 610.sup.4 cells/cm.sup.2 in an EC cell culture medium. After overnight incubation, the cells were treated with Advanced RPMI (A-RPMI 1640; Gibco-BRL) supplemented with 100 L-GlutaMAX (L-glu; Gibco-BRL) and 1% penicillin/streptomycin (P/S; Gibco-BRL) and 5 M CHIR99021 (glycogen synthase kinase-3 inhibitor; Stemgent, Lexington, Mass.), for 36-48 hours (for about 2 days). Thereafter, the cells were induced into IM by treatment with 100 ng/ml bFGF (Peprotech, Rocky Hill, N.J.) and 1 M retinoic acid (RA; Sigma, St. Louis, Mo.) for about 4 days. After about 2 days, the medium was replaced.

[0098] FIG. 1 illustrates a simplified diagram showing the method of specification from embryonic stem cells (ES) into intermediate mesoderm (IM) via mesendodem (ME).

[0099] FIG. 2 shows a day-2 (D2) phase change image of mESC treated with CHIR, and day-4, 5, and 6 (D4, D5, D6) phase change images of mESC treated with bFGF and RA (CHIR: CHIR99021).

[0100] As shown in FIG. 2, on day 2 after mESC was treated with CHIR99021 (D2), it was confirmed that the shape of the cell clusters did not change. In addition, after treatment with bFGF and RA for 4 days, 5 days, and 6 days (D4, D5, D6), it was confirmed that the cells changed and proliferated and the number of the attached cells was increased.

[0101] When the specification was progressed from mESC to IM, the expression of IM marker genes Pax2, Osr1, Lhx2 and Wt1 and the expression of pluripotent marker gene Oct4 were confirmed by RT-PCR analysis. For RT-PCR, total RNA was isolated from cells at each step using TRIzol Reagent (Invitrogen) and quantified using NANODROP 2000 UV/Ms Spectrophotometer (Thermo Scientific, Waltham, Mass.). RT-PCR was performed using First Strand cDNA Synthesis kit (Takara Bio, Shiga, Japan) and AccuPower PCR premix (Bioneer, Deajeon, Korea) according to the manufacturers instructions. All PCR products were isolated by 2% agarose gel electrophoresis. Primer sets used are shown in Table 1 below.

TABLE-US-00001 TABLE1 gene Fowarddirectionprimer Reverseprimer Oct4 5-TGTGGACCTCAGGTTGGA 5-TTTCATGTCCTGGGAC CT-3 TCCTC-3 Pax2 5-CTGTTTCCAGCGCCTCTA 5-GACGCTCAAAGACTCG AC-3 ATCC-3 Osr1 5-TTCGTTTGCAAGTTCTGT 5-TGTAGCGTCTTGTGGA GG-3 CAGC-3 Lhx1 5-CAGTGTCGCCAAAGAGA 5-TCAACGTCTCCAGTTG ACA-3 CTTG-3 Wt1 5-CCAGTGTAAAACTTGTCA 5-TGGGATGCTGGACTGT GCGA-3 CT-3

[0102] FIGS. 3 and 4 show the results of RT-PCR and real time PCR confirming mRNA expression levels of Oct4, Pax2, Osr1, Lhx2, and Wt1 on days 2, 4, 5, and 6 (D2, D4, D5, and D6) while mESC was induced into IM. As shown in FIGS. 3 and 4, while mESC was specified into IM, no expression of IM marker was detected in cells cultured with CHIR99021 for 2 days. However, after 2 days of treatment with bFGF and RA, expressions of Pax2, Lhx1 and Wt1 were detected, and Osr1 was not observed on day 2 of treatment with bFGF and RA (D4). After 4 days of treatment with bFGF and RA (D6), it was confirmed that all IM markers were expressed in the cells.

[0103] In addition, the expression of PAX2 and LHX1, which are IM markers, in mESC-induced IM were confirmed by immunofluorescent staining. Specifically, the analyte cells were fixed with 4% paraformaldehyde (PFA, Biosesang, Gyeonggi-do, Korea) in PBS at a temperature of 4 C. and permeated for 5 minutes with 0.1% Triton X-100 (Sigma) in PBS. Then, the cells were blocked with a blocking solution (DAKO North America Inc., Carpinteria, Calif.) at room temperature for 1 hour and incubated with primary antibody overnight at a temperature of 4 C. Secondary antibodies were incubated for 1 hour at room temperature, and the antibodies and dilutions used herein are as follows: LHX1 (1:100; Santa Cruz), PAX2 (1:100; Thermo Scientific), GATA4 (1:100; SantaCruz), and FSHR (1:100, Santa Cruz). Secondary antibodies used were as follows: Alexa-488, Alexa-594, and Alexa-488 (1:200, Life Technologies). Primary antibodies used for immunohistochemistry were as follows: GFP (1:100; Abcam, Boston, Mass.) and GATA4 (1:100; Santa Cruz). Secondary antibodies used were as follows: Alexa-488 and Alexa-594 (1:100, Life Technologies). After all treatments immunofluorescence images were obtained using a confocal microscope (Carl Zeiss LSM 880, Oberkochen, Germany).

[0104] FIG. 5 shows the results of immunofluorescence staining for IM markers PAX2 and LHX1 in mESC-induced IM.

[0105] As shown in FIG. 5, all IM markers were detected in the mESC-induced IM. Therefore, it was confirmed that the above-described treatment conditions can induce an IM exhibiting IM characteristics from mESC.

Example 2. Differentiation from mESC-Induced IM to Sertoli-Like Cells (SLC)

[0106] In order to differentiate the mESC-induced IM to SLC as in Example 1, the cells at the stage of IM were treated, for about 6 days, with 100 ng/ml bFGF, 100 ng/ml FGF-9 (Peprotech), 500 ng/ml prostaglandin D2 (PGD2, Santa Cruz Biotechnology, Dallas, Tex.), 10 ng/ml glia cell line-induced neurotrophic factor (GDNF; R&D, Minneapolis, Minn.), 10 ng/ml follicle stimulating hormone (FSH; Sigma), and 100ITS (insulin-transferrin-selenium; Invitrogen, Grand Island, N.Y.). Medium was exchanged every 2 days. The cell morphology was confirmed 6 to 7 days after the deriving.

[0107] FIG. 6 shows a schematic view illustrating a method of differentiating mESC-induced IM into SLC.

[0108] FIG. 7 shows phase change images of SLCs 3 days after differentiation from IM (D3 SLCs) and 7 days after differentiation from IM (D7 SLCs), and 5-day old mouse SCs, which are immature mouse SCs.

[0109] As shown in FIG. 7, after differentiation of IM to SLC, it was confirmed that, like immature mouse Sertoli cells, induced SLC formed a cord-like structure.

[0110] In addition, the experiment was performed as follows to determine whether the induced SLC as described above can easily form a tube-like structure. Immature Sertoli cells obtained from 5 day-old mouse (5 d mouse SCs) and SLC induced from ESCs were cultured for about 48 hours in a differentiation medium on matrigel (the same as the medium for SLC differentiation). As a control, cells cultured in 10% FBS were used. The morphologies of the test group and the control were used to compare the cord-like structures, and the results thereof are shown in FIG. 7.

[0111] As shown in FIG. 8, it was confirmed that, when cultured in the differentiation medium on Matrigel, cells, like 5 day-old mouse Sertoli cells, formed cell aggregates after about 48 hours and formed a 3D hollow cord-like structure. The 3D cord-like structure was hollow, like a tube, and produced a very uniform hexagonal alignment in the Matrigel. On the other hand, when cultured in 10% FBS, it was confirmed that the cells did not form the hollow cord-like structure.

[0112] Therefore, it was confirmed that, when cells were cultured in SLC differentiation medium on Matrigel, a web-like structure was formed. Since the web-like structure indicates the possibility of tube formation, it was confirmed that SLC induced from IM can easily form the tube-like structure.

[0113] In addition, in order to confirm whether SLC induced from IM has the characteristics of Sertoli cells, mRNA levels of Wt1, Sox9, Sf1, Gata4, Fshr, and Scf genes, which are the markers of Sertoli cells, and Oct4 gene, which is a pluripotency marker gene, were identified by RT-PCR and real time PCR. Information on the primer sets used for RT-PCR and real time PCR is shown in Table 2 below.

TABLE-US-00002 TABLE2 Gene Forwardprimer Reverseprimer Sox9 5-CACAAGAAAGACCACC 5-GGACCCTGAGATTGCCC CCGA-3 AGA-3 Sf1 5-AGAAGTTTCTGAGAGC 5-TACGAATAGTCCATGCCC CCGC-3 GC-3 Gata4 5-CTGGCCAGGACTGCCG- 5-GGTTGCTCCAGAAATCGT 3 GC-3 Fshr 5-AATCCGTGGAGGTTT 5-AGCACAAATCTCAGTTCA TCGCT-3 ATGGC-3 Scf 5-GAAGACACAAACTTGG 5-CATCCCGGCGACATAGTT ATTATCACT-3 GA-3

[0114] FIGS. 9 and 10 are the graphs showing the results of RT-PCR and real time PCR confirming mRNA expression levels of Oct4, Wt1, Sox9, Sf1, Gata4, Fshr, and Scf in mES cells, induced SLC, and SCs. As shown in FIGS. 9 and 10, sertoli cell markers were not expressed in mES, whereas expressions of sertoli cell markers Wt1, Sox9, Sf1, Gata4, Fshr and Scf in induced SLC were at the level similar to those of Sertoli cells.

[0115] Therefore, since the SLC induced from IM expresses sertoli cell markers, it was confirmed that the cells were SLC having the characteristics of Sertoli cells.

[0116] In addition, the expression of serotoli cell markers GATA4 and FSHR in induced SLC was confirmed by immunofluorescent staining. As a control, 5 day-old mouse Sertoli cells (5 d mouse SCs) were used.

[0117] FIG. 11 shows immunofluorescence staining results for SC markers GATA4 and FSHR in SLC induced from IM.

[0118] As shown in FIG. 11, since the sertoli cell markers were detected in SLC induced from IM, it was confirmed that SLC induced from IM exhibited the characteristics of the Sertoli cells.

[0119] Therefore, it was confirmed that ESC-induced SLC was differentiated to SLC under the above conditions and that characteristics of SLC were similar to those of immature Sertoli cells obtained from 5 day-old mouse.

Example 3. Isolation, Purification and Characterization Identification of Sertoli Cells of Induced SLC

[0120] 3.1 Isolation and Purification of Induced SLC

[0121] As shown in FIG. 11, the weak expression of Oct4 mRNA, which is a pluripotent marker in SLC, indicates that ESC that is undifferentiated or SLC that is in differentiation remains in differentiated SLC. Therefore, in order to isolate and purify differentiated SLC from the culture, MACS using an antibody against FSHR, which is a testicle sertoli cell marker, was performed.

[0122] Specifically, differentiated SLC 110.sup.7, was trypsinized, collected, and the anti-FSHR-biotin antibody (1:20, Bioss, Woburn, Mass.) was Incubated in 100 l MACS solution (Miltenyi Biotec, Gladbach) for 30 minutes at room temperature. Unbound anti-FSHR-biotin antibody was washed and removed by centrifugation twice at 300g for 10 min after the addition of 1 ml to 2 ml of buffer. Cell pellets were resuspended in 80 l buffer, and 20 l anti-biotin microbeads (UltraPure, Miltenyi Biotec) were added thereto and mixed well, followed by incubation at a temperature of 4 C. for 15 minutes. Cells were then washed with 2 ml 0.5% BSA (Sigma) in PBS buffer and centrifuged at 300g for 10 minutes to remove excess beads from the solution. The wash solution is then processed and, according to the manufacturer's guidelines, the pellets were resuspended in 500 l buffer at maximum column capacity and the suspension was placed on a pretreated LD column (Miltenyi Biotec) mounted on a MACSMidi magentic cell isolator (Miltenyl Biotec). The column was washed twice with 2 ml buffer to remove unmarked cells. After removing the column from the magentic isolator, the isolated cells were eluted with 1 ml buffer to collect purified, isolated SLC.

[0123] 3.2 Characterization of Cells after Isolation and Purification of Induced SLC

[0124] In order to confirm that the induced SLC was purified and isolated by the above method, immunostaining was performed on the sertoli cell markers FSHR and GATA4. FIG. 12 shows an image showing the results of immunostaining of SLC before and after purification and isolation with FSHR and GATA4.

[0125] As shown in FIG. 12, it was confirmed that the ratio of FSHR and GATA4-double positive SLC increased after isolation as compared with before isolation and purification.

[0126] In addition, flow cytometric analysis was further performed as follows. SLC before and after isolation were collected and fixed with 4% paraformaldehyde for 4 minutes at room temperature. After washing, the cells were permeated with cooled 90% methanol for 10 minutes. Cells were then blocked with 0.5% BSA (Sigma)/PBS for 30 minutes at room temperature and cultured with primary antibody. To assess the efficiency of FSHR MACS, cells were incubated with anti-rabbit FSHR antibody (Santa Cruz) and anti-mouse GATA4 antibody at a temperature of 4 C. for 1 hour. Secondary antibodies were detected by incubation using APC-conjugated (Life technology) and PE-conjugated antibodies at a temperature of 4 C. for 1 hour. Control cells were not treated with the primary antibody. Cells were stored on ice in the dark condition until analysis, using Becton DicKinson FACS IV Calibur (Becton Dickinson, San Jose, Calif.). At least 5,000 or 10,000 events were collected for each sample.

[0127] FIG. 13 shows a graph of the flow cytometry results before and after isolation.

[0128] As shown in FIG. 13, it was confirmed that the FSHR-positive SLC ratio (90.05.9%) after isolation was significantly greater than the FSHR-positive SLC ratio (15.42.0%) before isolation. In addition, it was confirmed that most of the detected cells were FSHR and GATA4-double positive SLC.

[0129] In addition, mRNA expression levels of Oct4, Wt1, Sox9, Gata4, and Fshr genes in FSHR-positive SLC and FSHR-negative SLC were confirmed by real time PCR and RT-PCR. Specifically, real time PCR was performed in such a manner that total RNA was extracted from each cell using the method as described above, and for quantification of gene expression levels, in Bio-Rad CFX96 real-time PCR, iQ\SYBR Green supermix (Bio-Rad Laboratories, Alfred Nobel Drive Hercules, Calif.) was used at the final concentration of 25 ng cDNA. The expression levels of each gene were normalized to the levels of Gapdh using the Ct method and expressed as relative to mESC. Primers used for real time RT PCR are shown in Table 1 and Table 2. The results of real time RT PCR are shown in FIG. 14, and the results of RT-PCR are shown in FIG. 15.

[0130] As shown in FIGS. 14 and 15, it was confirmed that mRNA expression levels of the sertoli cell markers Wt1, Sox9, Gata4, and Fshr were much higher in FSHR-negative SLC (FSHR ()) than in FSHR-positive SLC (FSHR (+)).

[0131] Therefore, it was confirmed that FSHR-positive SLC expresses the characteristics of mature Sertoli cells.

Example 4. Functional Analysis of Induced SLC

[0132] 4.1 Identification of Immunological Features of Induced SLC

[0133] In order to confirm the immunological function of SLC induced from the present disclosure, Bululfan-treated ICR mouse was as the receptor animal. The receptor mice was anesthetized with avertin and the cell suspension (MACS-isolation cells) expressing EGFP described in the above example was injected in an amount of less than 10 l at 110.sup.5 cells/testicle, thereby transplanting cells into the seminiferous tubule via the export duct.

[0134] FIG. 16 shows images of a testicle and seminiferous tubule of a mouse of which EGFP expression was confirmed by ultraviolet (UV) light after SLC transplantation.

[0135] As shown in FIG. 16, it was confirmed that EGFP was expressed in the testicle and seminiferous tubule of the Busulfan-treated receptor mouse.

[0136] Therefore, it was confirmed that the SLC was well transplanted into the mouse testicle, and that SLC that was drived in vitro can be transplanted in vivo.

[0137] FIG. 17 shows the results of immunofluorescence of testicles stained with anti-GATA4 (red) and anti-GFP (green) antibodies 1 and 7 days after SLC transplantation.

[0138] As shown in FIG. 17, it was confirmed by immunohistochemical analysis that a few EGF-positive SLCs were observed at the base of seminiferous tubule of receptor mound and co-expressed with SC marker GATA4. On day 1 after transplantation, clusters were detected in the seminiferous tubule of the testicle of the receptor mouse (top panel). On day 7 after transplantation, transplanted cells were located at the base of the seminiferous tubule of the receptor mouse (lower panel).

[0139] Therefore, it was confirmed that ESC-induced SLC can be transplanted into the seminiferous tubule of the receptor and, after transplantation, like Sertoli cells, located at the base of the seminiferous tubule and can functionally replace Sertoli cells.

[0140] 4.2 Identification of Phagocytosis Activity of Induced SLC

[0141] In order to confirm the phagocytic activity of induced SLC according to the present disclosure, in vitro experiments were performed on Sertoli cells and induced SLC as follows.

[0142] With respect to adult SC, 5 day-old SC, induced SLC, FSHR-positive SLC, FSHR-negative SLC, and mouse embryonic fibroblasts (MEF), the uptake of fluorescein labeled E. coli was measured using a commercially available kit (Vybrant Phagocytosis Assay kit, Molecular Probes, Eugene, Oreg.). Each cell was seeded in a 4-well plate at a concentration of 210.sup.5 cells/well in s SLC differentiation medium at 5% CO.sup.2 and at a temperature of 37 C. After 12 hours of inoculation, each cell was washed with PBS and incubated overnight in a SLC differentiation medium containing E. coli labeled with fluorescein. At the end of the incubation period, each cell was washed twice to remove E. coli that did not undergo extracellular phagocytosis, and a solution containing 100 ng/ml Hoechst (for staining cell nuclei; Thermo Scientific) in PBS was added in an amount of 0.5 ml to each well, followed by the incubation at a temperature of 37 C. for 15 minutes. Each cell was then washed twice with PBS. An image of each cell was obtained using a confocal microscope (Carl Zeiss LSM 880). Phagocytosis activity was measured with respect to cells having GFP particles/whole cells by using ImageJ (NIH, Bethesda, Md.) software.

[0143] FIG. 18 shows images showing phagocytosis activity of FSHR-positive SLC (FSHR (+)) and FSHR-negative SLC (FSHR ()). Blue: Hoechst-stained nucleus; Green: fluorescent microbeads; Red: induced SLC, derived from RFP-transplanted mESCs.

[0144] FIG. 19 shows a graph comparing phagocytosis activities of adult SC, 5-day old SC, FSHR-positive SLC, FSHR-negative SLC and MEF. Phagocytosis activity was calculated with respect to cells/whole cells (meanSEM) containing fluorescent microbeads, with different alphabets representing P<0.05.

[0145] As shown in FIGS. 18 and 19, the phagocytosis activity of FSHR-positive SLC was significantly greater than MEF cells (somatic control) and FSHR-negative SLC, similar to that of the adult Sertoli cells.

[0146] 4.3 Identification of CD4.sup.+ T Cell Proliferation Activity of Induced SLC (CD4+ T Cell Proliferation Assay)

[0147] In order to confirm CD4.sup.+ T cell proliferation activity through CFSE of induced SLC according to the present disclosure, the induced SLC and other cells were compared and analyzed as follows.

[0148] Splenocytes were isolated from 6 to 8 week-old male mouse, and erythrocytes were removed by adding erythrocyte lysate (Sigma). For isolation of CD4.sup.+ T cells, the cells were isolated using the CD4 T cell isolation kit (Miltenyi Biotecn) according to the manufacturer's method. Isolated CD4.sup.+ T cells were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSC, BD bioscience). 1.2 M of CFSE was labeled at a temperature of 37 C. for 10 minutes and washed three times at room temperature. CFSE-labeled CD4.sup.+ T cells were cocultured with splenocytes, human bone marrow induced mesenchymal stem cells (hBM-MSC, Lonza), adult SC, and induced SLC (FSHR (+)) at a 5:1 ratio for 5 days. To induce the proliferation of CD4.sup.+ T cells, 50 ng/ml of phorbol myristate acetate (PMA, Sigma) and 1 M of ionomycin (Sigma) were co-cultured in culture. After co-culture, proliferative activity of CD4.sup.+ T cells was measured by flow cytometry (BD bioscience).

[0149] FIG. 20 is a histogram confirming CD4.sup.+ T cell proliferation activity through CSFE in splenocytes, hBM-MSC, adult SC, and induced SLC.

[0150] FIG. 21 is a graph showing the average value of the CD4.sup.+ T cell proliferation activities which were measured three or more times through CSFE in splenocyte, hBM-MSC, adult SC, and induced SLC. Different alphabets represent P <0.05.

[0151] As shown in FIGS. 20 and 21, the CD.sup.4+ T cell proliferative activity of induced SLC was significantly lower than in a group treated with PMA and ionomycin to induce proliferation, splenocytes and hBM-MSC, and was similar to that of adult SC.

[0152] In addition, to determine whether the SLC according to the present disclosure exhibits sertoli cell-like functions, the mRNA expression level of Transferrin, which is a marker of Sertoli cells, and the mRNA expression level of TGF-1, Il-6, Fas-L and Clusterin, which are immunomodulatory genes, were confirmed in un-isolated SLC, FSHR-positive SLC (FSHR (+) SLCs), and an adult testicle by real time PCR and RT-PCR. Primer sets used for real time PCR and RT-PCR are shown in Table 3 below.

TABLE-US-00003 TABLE3 Gene Forwardprimer Reverseprimer TGF-1 5-CCGCAACAACGCCA 5-TGCCGTACAACTCCAG TCTATG-3 TGAC-3 Transferrin 5-TCTTCTCGGGCAGT 5-CATGAGAAGGGATCCG TGTGTC-3 AGCC-3 II-6 5-AGCCAGABTCCTTC 5-TGGTCTTGGTCCTTAG AGAGAGA-3 CCAC-3 Fas-L 5-GAACTGCGAGAACT 5-ACTCCAGAGATCAGAG CCGTGA-3 CGGT-3 Clusterin 5-GGGTGTACTTGAGC 5-TCCTTGGAATCTGGAG AGAGC-3 TCCGGT-3

[0153] FIG. 22 shows the results of RT-PCR confirming mRNA expression levels of TGF-1, Transferrin, IL-6, Fas-L and Clusterin in un-isolated SLC, FSHR-positive SLC, and adult testicles. As shown in FIG. 22, it was confirmed that Clusterin, Il-6 and TGF-1 were highly expressed in FSHR-positive SLC and adult testicle tissues. In addition, the expression of Transferrin was detected in FSHR-positive SLC and adult testicle tissues.

[0154] Therefore, it was confirmed that due to the method according to the present disclosure, SLC induced in mESC has an immunosuppressive function and functional characteristics of mature Sertoli cells.

[0155] Statistical Analysis

[0156] Unless otherwise indicated, all data represent at least three independent experiments. The results were expressed as meanSEM. Statistical significance was assessed using one-way ANOVA and Tukey test.