Patent ID: 12234481

DETAILED DESCRIPTION OF THE EMBODIMENTS

The following examples and experimental examples are provided to further illustrate the present disclosure, and shall not be construed as a limitation to the present disclosure. Moreover, the examples do not include detailed descriptions of traditional methods.

Example 1

Screening of Candidate Markers for dNK Cells

In the statistical calculations in this example and the following examples, different statistical modes of the software SPSS 22.0 were selected to calculate the p value according to requirements for evaluation of the significance of differences between 2 groups, differences among 3 or more groups, or difference between ratios.

Decidual tissues were collected from 10 healthy cases undergoing early pregnancy termination due to non-medical reasons (normal group) and 5 cases undergoing early pregnancy termination due to spontaneous abortion (abortion group), and NK cells were isolated by fluorescence-activated cell sorting (FACS) with reference to the literature [Fu B, et al. Immunity, 2017, 47 (6): 1100-1113.e6.]. For example: The decidual tissue was digested with 1 mg/mL collagenase IV (Sigma-Aldrich) and 0.01 mg/mL DNase I (Shanghai Sangon) for 1 h, then lymphocytes were obtained by Percoll (GE Healthcare) density gradient centrifugation and then cultivated on a petri dish at 37° C. for 2 h to remove stromal cells and macrophages, and NK cells were isolated by FCM. The CD56 antibody, CD3 antibody, and CD14 antibody were first used to preliminarily sort out NK cells, and then the CD16 antibody and CD49a antibody were used to further sort out dNK cells, which were dNK cells with a phenotype of CD56brightCD16−CD49a+(CD56brightCD16−CD49a+CD3−CD14−). NK cells obtained in the normal group and the abortion group were lysed, a protein concentration was determined by the Bradford method, and lysates of the two groups were subjected to protein expression analysis by the iTRAQ-nano-HPLC-MS/MS method, thus determining different membrane surface marker expression levels on the groups of NK cells. The methods could refer to the literature [Jiang, Hong-Lin, et al. Cancer research 76.4 (2016): 952-964.]. According to clustering statistical analysis of membrane surface markers, the expression of CD39 (UniProtKB: P49961), CD27 (UniProtKB: P26842), CD160 (UniProtKB: 095971), and TIGIT (UniProtKB: Q495A1) on dNK cells of the normal group was significantly higher than that on dNK cells of the abortion group.

Example 2

Preparation of dNK Cells and dNK Cell Subsets

A decidual tissue collected from a case undergoing early pregnancy termination due to non-medical reasons was used to prepare dNK cells, which was implemented according to the method described in Example 1. Briefly: The decidual tissue was digested with 1 mg/mL collagenase IV (Sigma-Aldrich) and 0.01 mg/mL DNase I (Shanghai Sangon) for 1 h, and then lymphocytes were obtained by Percoll (GE Healthcare) density gradient centrifugation. The lymphocytes were cultivated at 37° C. for 2 h on a petri dish to remove stromal cells and macrophages, and then NK cells were isolated by FCM. dNK cells with a phenotype of CD56brightCD16−CD49a+TIGIT+were obtained. Antibody magnetic beads were used to further sort out CD56brightCD16−CD49a+CD39k-positive dNK cell subset, CD56brightCD16−CD49a+CD27+-positive dNK cell subset, CD56brightCD16−CD49a+CD160+dNK cell subset, CD56brightCD16−CD49a+TIGIT+-positive dNK cell subject, and CD56brightCD16−CD49a+CD39+TIGIT+-positive dNK cell subset. The obtained cells could be directly tested, used, or cryopreserved. The peripheral blood was collected from the same volunteer, and NK cells were isolated according to the general method and used as control cells.

Example 3

Preparation of Exosomes Derived from dNK Cells and dNK Cell Subsets

The dNK cells and dNK cell subsets and control NK cells freshly isolated in Example 2 were cultivated in a serum-free 1640 medium for 24 h. The cells were removed by centrifugation, a culture supernatant was filtered through a 0.45 μm filter membrane and then centrifuged at 4° C. and 1,000 g for 10 min, and a resulting supernatant was collected; the collected supernatant was centrifuged at 4° C. and 2,000 g for 20 min, and a resulting supernatant was collected; the collected supernatant was centrifuged at 4° C. and 10,000 g for 30 min, and a resulting supernatant was collected; the collected supernatant was centrifuged at 110,000 g for 100 min, a resulting supernatant was discarded, and a resulting precipitate was resuspended with PBS; and a resulting suspension was centrifuged once again at 110,000 g for 100 min, a resulting supernatant was discarded, and a resulting precipitate was resuspended with a small amount of PBS and then filtered through a 0.45 μm filter membrane to obtain an exosome. The Bradford method was used to detect the total exosome protein (Bio-Rad Protein Assay Reagent). Obtained exosomes were lyophilized and stored at −80° C. The following six exosomes were obtained: CD56brightCD16−CD49a+-positive dNK cell-derived exosome, CD56brightCD16−CD49a+CD39+-positive dNK cell-derived exosome, CD56brightCD16−CD49a+CD27+-positive dNK cell-derived exosome, CD56brightCD16−CD49a+CD160+-positive dNK cell-derived exosome, CD56brightCD16−CD49a+TIGIT+-positive dNK cell-derived exosome, and CD56brightCD16−CD49a+CD3930TIGIT+-positive dNK cell-derived exosome. An exosome derived from the peripheral NK cells in Example 2 was used as a control exosome.

Example 4

Effects of the Exosomes to Enhance Endometrial Cell Viability, Reduce Endometrial Cell Damage, and Increase VEGF Expression

Non-pathological endometrial stromal cells were cultivated for 24 h, and each of the exosomes obtained in Example 3 was added, where a ratio of a mass of the exosome protein to a volume of the medium matrix was 0.02% (that is, a relative mass-to-volume ratio). The control exosome in Example 3 was used in the control group, and no exosome was used in the blank group. After the treatment was conducted for 48 h, stromal cells and media were sampled for analysis.

The cell viability of the stromal cells was determined by the PrestoBlue method (Thermo Fisher Scientific), and the determination was conducted 48 h after the treatment. The value was expressed as an average value (%) obtained after normalization relative to the control (Table 1).

TABLE 1Relative cell viability of stromal cellsAveragepGroup (exosome treatment group, 0.02%)valueSDvalueBlank (medium only)100.0014.63Control exosome101.664.55CD56brightCD16−CD49a+-positive dNK158.3724.99p <cell-derived exosome0.05CD56brightCD16−CD49a+CD39+-positive140.498.32p <dNK cell subset-derived exosome0.05CD56brightCD16−CD49a+CD27+-positive172.6924.81p <dNK cell subset-derived exosome0.05CD56brightCD16−CD49a+CD160+-positive159.9011.93p <dNK cell subset-derived exosome0.05CD56brightCD16−CD49a+TIGIT+-positive161.1418.66p <dNK cell subset-derived exosome0.05CD56brightCD16−CD49a+CD39+TIGIT+-positive179.3823.61p <dNK cell subset-derived exosome0.05

Conclusion: The exosomes derived from dNK cells and dNK cell subsets of the present disclosure have the ability to increase the uterine stromal cell viability, and can be used as a product for enhancing endometrial proliferation.

Further evaluation of a stromal cell damage level: A lactate dehydrogenase (LDH) detection kit was used to determine a cell damage level by colorimetry, such that cell damage could be quantified based on the determination of LDH activity in damaged cells in the medium. Increased cell membrane damage and cell lysis lead to an increase in LDH activity, which was proportional to the number of lysed cells. After the exosome treatment was conducted for 48 h, LDH activity was determined in the medium, and a value was expressed as an average value (%) obtained after normalization relative to the control (Table 2).

TABLE 2LDH activityAveragepGroup (exosome treatment group, 0.02%)valueSDvalueBlank (medium only)100.0015.75Control exosome103.357.65CD56brightCD16−CD49a+-positive dNK cell-67.777.11p <derived exosome0.05CD56brightCD16−CD49a+CD39+-positive63.547.64p <dNK cell subset-derived exosome0.05CD56brightCD16−CD49a+CD27+-positive49.017.10p <dNK cell subset-derived exosome0.05CD56brightCD16−CD49a+CD160+-positive36.125.86p <dNK cell subset-derived exosome0.05CD56brightCD16−CD49a+TIGIT+-positive49.507.33p <dNK cell subset-derived exosome0.05CD56brightCD16−CD49a+CD39+TIGIT+-positive32.715.08p <dNK cell subset-derived exosome0.05

Conclusion: The exosomes derived from dNK cells and dNK cell subsets of the present disclosure have the ability to alleviate membrane damage, and can be used as a product for enhancing stromal cell viability.

The potential enhancement effect of the exosomes on the VEGF expression of endometrial stromal cells was further investigated. After the exosome treatment was conducted for 48 h, the VEGF concentration in the medium was determined by ELISA. Results were shown in Table 3.

TABLE 3VEGF expressionVEGFpGroup (exosome treatment group, 0.02%)(pg/ml)SDvalueBlank (medium only)250.7724.63Control exosome244.3620.55CD56brightCD16−CD49a+-positive dNK cell-491.3738.32p <derived exosome0.05CD56brightCD16−CD49a+CD39+-positive666.5142.20p <dNK cell subset-derived exosome0.05CD56brightCD16−CD49a+CD27+-positive701.7097.36p <dNK cell subset-derived exosome0.05CD56brightCD16−CD49a+CD160+-positive998.6555.49p <dNK cell subset-derived exosome0.05CD56brightCD16−CD49a+TIGIT+-positive842.7689.50p <dNK cell subset-derived exosome0.05CD56brightCD16−CD49a+CD39+TIGIT+-positive572.3376.63p <dNK cell subset-derived exosome0.05

Conclusion: The exosomes derived from dNK cells and dNK cell subsets of the present disclosure can promote the expression of VEGF, which has the effect of enhancing endometrial angiogenesis.

Example 5

Promotion of the Exosomes on the Marker Expression on Endometrial Stromal Cells

Non-pathological endometrial stromal cells were cultivated for 24 h, and each of the exosomes obtained in Example 3 was added, where a ratio of a mass of the exosome protein to a volume of the medium matrix was 0.02% (that is, a relative mass-to-volume ratio). The control exosome in Example 3 was used in the control group. The stromal cells were further cultivated for 24 h in an incubator at 37° C. and 5% CO2, and then the ALDH positive rate and Ki67 positive rate were determined for the stromal cells by FCM. Results were shown in Tables 4 and 5.

TABLE 4ALDH positive rateALDHpositivepGroup (exosome treatment group, 0.02%)rateSDvalueBlank (medium only)3.650.74Control exosome3.150.66CD56brightCD16−CD49a+-positive dNK cell-20.642.09p <derived exosome0.05CD56brightCD16−CD49a+CD39+-positive28.152.87p <dNK cell subset-derived exosome0.05CD56brightCD16−CD49a+CD27+-positive22.142.75p <dNK cell subset-derived exosome0.05CD56brightCD16−CD49a+CD160+-positive30.454.31p <dNK cell subset-derived exosome0.05CD56brightCD16−CD49a+TIGIT+-positive20.763.21p <dNK cell subset-derived exosome0.05CD56brightCD16−CD49a+CD39+TIGIT+-positive21.242.39p <dNK cell subset-derived exosome0.05

TABLE 5Ki67 positive rateK167positivepGroup (exosome treatment group, 0.02%)rateSDvalueBlank (medium only)13.381.83Control exosome12.572.15CD56brightCD16−CD49a+-positive dNK cell-37.205.92p <derived exosome0.05CD56brightCD16−CD49a+CD39+-positive38.635.50p <dNK cell subset-derived exosome0.05CD56brightCD16−CD49a+CD27+-positive44.293.17p <dNK cell subset-derived exosome0.05CD56brightCD16−CD49a+CD160+-positive42.343.89p <dNK cell subset-derived exosome0.05CD56brightCD16−CD49a+TIGIT+-positive38.635.50p <dNK cell subset-derived exosome0.05CD56brightCD16−CD49a+CD39+TIGIT+-positive46.472.75p <dNK cell subset-derived exosome0.05

The results show that the exosomes derived from dNK cells and dNK cell subsets of the present disclosure have a very strong ability to maintain stemness and stimulate proliferation of stromal cells.

Example 6

Treatment of Endometrial Injury with the Exosomes

Patients had an endometrial thickness of less than 8 mm due to induced abortion, dilatation and curettage, infection, and other factors, which was clinically diagnosed as thin endometrium. Anti-infection and other treatments were given to the patients, which showed no effects. The patients were administered with a composition with the exosomes (exosomes derived from dNK cells and dNK cell subsets) prepared in Example 3 as an active ingredient at a dosage of 10 mg/kg. The composition could be administered by intravenous infusion or intrauterine perfusion, for example. The composition was administered to the patient one or more times to promote the increase in endometrial thickness.

Example 7

Effect of the Exosomes on Decidual Dendritic Cells (dDCs)

A decidual tissue was collected from a person undergoing pregnancy termination due to non-medical reasons, and DCs (CD1c positive) were isolated and sorted with reference to the literature (Guo P F, et al. Blood, 2010, 116 (12): 2061-2069). The DC cells were divided into a negative control group (treated with the control exosome described in Example 3, at a mass-to-volume ratio of 0.02%), treatment groups (treated with the exosomes derived from dNK cells and dNK cell subsets described in Example 3, at a mass-to-volume ratio of 0.02%), an LPS treatment group (100 ng/ml), and a blank group (without exosome). After the cells were cultivated for 48 h, the interleukin 10 (IL-10) and tumor necrosis factor α (TNF α) levels in a cultivation system were detected by methods described in the literature (Guo P F, et al. Blood, 2010, 116 (12): 2061-2069). Results showed that the exosomes significantly increased the IL-10 level, but did not increase the TNFα level (Tables 6 and 7). These results further confirmed that the exosomes derived from dNK cells and dNK cell subsets could exert immune tolerance through DCs.

TABLE 6IL-10 contentIL-10pGroup (exosome treatment group, 0.02%)(pg/ml)SDvalueBlank (medium only)45.084.35Control exosome49.555.15CD56brightCD16−CD49a+-positive dNK cell-542.7984.21p <derived exosome0.05CD56brightCD16−CD49a+CD39+-positive520.5473.93p <dNK cell subset-derived exosome0.05CD56brightCD16−CD49a+CD27+-positive500.9534.03p <dNK cell subset-derived exosome0.05CD56brightCD16−CD49a+CD160+-positive595.8495.13p <dNK cell subset-derived exosome0.05CD56brightCD16−CD49a+TIGIT+-positive483.6335.80p <dNK cell subset-derived exosome0.05CD56brightCD16−CD49a+CD39+TIGIT+-positive496.9563.74p <dNK cell subset-derived exosome0.05LPS391.7547.56p <0.05

TABLE 7TNFα contentTNFαpGroup (exosome treatment group, 0.02%)(pg/ml)SDvalueBlank (medium only)27.763.43Control exosome36.554.11CD56brightCD16−CD49a+-positive dNK cell-13.821.17p >derived exosome0.05CD56brightCD16−CD49a+CD39+-positive39.364.39p >dNK cell subset-derived exosome0.05CD56brightCD16−CD49a+CD27+-positive32.114.73p >dNK cell subset-derived exosome0.05CD56brightCD16−CD49a+CD160+-positive35.874.20p >dNK cell subset-derived exosome0.05CD56brightCD16−CD49a+TIGIT+-positive21.142.99p >dNK cell subset-derived exosome0.05CD56brightCD16−CD49a+CD39+TIGIT+-positive32.832.33p >dNK cell subset-derived exosome0.05LPS1222.6762.50p <0.05

Example 8

Therapeutic Effect of the Exosomes on Spontaneous Abortion Models

CBA/J female mice and DBA/2J male mice were used to establish stress abortion models, which were classic research models for maternal-fetal immune tolerance disorders. The establishment method, experimental method, and observation time points could be seen in the literature (Blois S M, et al. Nature Medicine, 2007, 13 (12): 1450-1457). CBA/J female mice were divided into a negative control group, a stress group, a control group, and treatment groups before being raised together. The treatment groups were intravenously administered with the exosomes derived from dNK cells and dNK cell subsets of the present disclosure (150 μg/mouse) once every 3 days, with a total of 3 administrations. The control group was administered with the control exosome at the same dosage in the same administration route. The mice were raised together 3 days after the first administration. The mice were raised separately immediately after the pregnancy was determined by vaginal plug (effective n=10).

The experimental results (Table 8) showed that the abortion rate of the treatment group was significantly lower than that of the stress abortion group, indicating that the exosomes derived from dNK cells and dNK cell subsets have prominent therapeutic effects.

TABLE 8Analysis of embryo absorption rate(abortion) of mice in each groupp value(relative toEmbryostress +absorp-controlGrouption rateSDcells)Blank control group9.469.41Stress + control exosome38.819.92CD56brightCD16−CD49a+-positive dNK cell-13.7614.36p < 0.05derived exosomeCD56brightCD16−CD49a+CD39+-positive12.3810.27p < 0.05dNK cell subset-derived exosomeCD56brightCD16−CD49a+CD27+-positive17.309.03p < 0.05dNK cell subset-derived exosomeCD56brightCD16−CD49a+CD160+-positive8.049.12p < 0.05dNK cell subset-derived exosomeCD56brightCD16−CD49a+TIGIT+-positive12.8610.96p < 0.05dNK cell subset-derived exosomeCD56brightCD16−CD49a+CD39+TIGIT+-11.6213.26p < 0.05positive dNK cell subset-derived exosome

Example 9

Effect of the Exosomes Derived from dNK Cells and dNK Cell Subsets on Helper T Cells

The para-aortic lymph nodes were collected from the mice in the control group, the stress group, the control exosome group, and the NK cell-derived exosome treatment groups in Example 8, and the Foxp3-positive helper T cell levels in the lymph nodes were detected. The collection method and detection method could be seen in the literature (Kim B J, et al. Proceedings of the National Academy of Sciences, 2015, 112 (5): 1559-1564). Results showed that the treatment with the exosomes derived from dNK cells and dNK cell subsets could effectively increase the Foxp3-positive helper T cell level (Table 9).

TABLE 9Foxp3% expression analysis for mice in each groupPer-p valuecentage(relativeof Foxp3-to stress +positivecontrolGroupTreg cellsSDcells)Blank control group42.855.78Stress + control exosome8.390.80CD56brightCD16−CD49a+-positive dNK35.633.11p < 0.05cell-derived exosomeCD56brightCD16−CD49a+CD39+-positive28.461.51p < 0.05dNK cell subset-derived exosomeCD56brightCD16−CD49a+CD27+-positive21.152.22p < 0.05dNK cell subset-derived exosomeCD56brightCD16−CD49a+CD160+-positive30.934.97p < 0.05dNK cell subset-derived exosomeCD56brightCD16−CD49a+TIGIT+-positive32.343.08p < 0.05dNK cell subset-derived exosomeCD56brightCD16−CD49a+CD39+TIGIT+-28.723.28p < 0.05positive dNK cell subset-derived exosome

Example 10

Treatment of Mouse Endometrial Injury Models with the Exosomes Derived from dNK Cells and dNK Cell Subsets

Establishment of animal endometrial injury models (C57 mice): 8-week-old female mice were divided into groups, each with 10 mice, and the double (infection+mechanical) damage method was used to establish the endometrial injury models. Specifically, the mice were anesthetized, a longitudinal incision of about 2 cm in the middle of the lower abdomen was provided to make a 0.5 cm longitudinal incision at a lower part ⅓ from the middle of the uterus; then an endometrial curette was used to curette the middle and upper segments of the uterine cavity; when the concave-convex feeling disappeared and the walls showed the roughness touch, the curettage was stopped; lipopolysaccharide cotton threads were left in the uterine cavity after curettage, and the abdominal incision was sutured; and the lipopolysaccharide cotton threads were taken out 48 h later. After the modeling was completed, the following groups were set: a blank control group (sham operation group); a group injected only with normal saline (NS) (model); model+control exosome; and NK cell-derived exosome treatment groups. The treatment groups were intravenously administered with the exosomes derived from dNK cells and dNK cell subsets of the present disclosure (150 μg/mouse) once every 3 days, with a total of 3 administrations. The female mice were mated with male mice after 3 estrous cycles. 1 month later, samples were collected for HE staining and Masson staining to evaluate the function of the endometrial tissue. 3 months later, pregnancy results were evaluated for the mice. Results: The histological function evaluation 1 month after the operation showed that, compared with the control group, the groups administered with the exosomes derived from dNK cells and dNK cell subsets had a significantly-reduced fibrosis degree; and compared with the control group, the exosome treatment groups had a larger number of secretory glands. The evaluation of pregnancy results showed that the groups administered with the exosomes derived from dNK cells and dNK cell subsets had a conception rate higher than that of the control exosome group. The results were shown in Table 10.

TABLE 10Pregnancy result analysis of mice in each groupp valueConcep-(relative toGrouption ratecontrol cells)Blank control group100%Model group20%Model group + control exosome20%CD56brightCD16−CD49a+-positive dNK cell-50%p < 0.05derived exosomeCD56brightCD16−CD49a+CD39+-positive60%p < 0.05dNK cell subset-derived exosomeCD56brightCD16−CD49a+CD27+-positive70%p < 0.05dNK cell subset-derived exosomeCD56brightCD16−CD49a+CD160+-positive60%p < 0.05dNK cell subset-derived exosomeCD56brightCD16−CD49a+TIGIT+-positive70%p < 0.05dNK cell subset-derived exosomeCD56brightCD16−CD49a+CD39+TIGIT+-positive60%p < 0.05dNK cell subset-derived exosome

Example 11

Effect of the Exosomes on Fertilized Eggs

C57BL/6J female mice (21 to 27 weeks old, body weight: 20.0 g to 24.5 g) were used to collect fertilized eggs. C57BL/6J male mice (32 to 38 weeks old, body weight: 31.0 g to 35.5 g) were used for mating. According to the conventional method of inducing excessive ovulation, 5 U (unit) of equine chorionic gonadotropin (eCG) was administered intraperitoneally to each female mouse, and 45 h to 48 h later, 5 U (unit) of human chorionic gonadotropin (HCG) was administered intraperitoneally to each female mouse. Each female mouse was mated with each of the above-mentioned male mice immediately after the administration of HCG.

The next day, it was determined whether the female mice after mating had a milky-white resinous vaginal plug, and the fallopian tubes were collected from female mice confirmed to have the vaginal plug. The collected fallopian tubes were statically placed in NS (0.9% (w/v) NaCl) for about 15 min and then transferred into an M16 medium added with about 300 μg/mL hyaluronidase (manufactured by Sigma-Aldrich) to remove cumulus cells. The fallopian tubes were cut, and fertilized eggs were taken out and statically incubated in a CO2incubator for 5 min to 10 min at 37° C. Then the fertilized eggs obtained after cumulus cells were removed were recovered, and washed with an M16 medium without hyaluronidase to remove the hyaluronidase. The fertilized eggs obtained after cumulus cells were removed were statically placed in a CO2incubator at 37° C.

In addition, drops were made with 100 μL of M16 medium in a 35 mm petri dish, and the drops were overlaid with mineral oil (produced by Sigma-Aldrich), and added with the exosomes derived from dNK cells and dNK cell subsets and the control exosome in Example 3, with a mass-volume ratio of 0.02%. The petri dish was statically placed in a CO2incubator at 37° C. 30 of the fertilized eggs obtained after cumulus cells were removed were transferred on the drops, and cultivated in vitro at 37° C. in a CO2incubator.

24 h, 48 h, 72 h, and 96 h after the beginning of in vitro cultivation in the drops (0 h), the developmental stage of each embryo was observed through a stereoscopic microscope, and the number and development rate of embryos developing normally were calculated. Specifically, for embryos developing normally at each stage, 24 h after the beginning of cultivation, the number of eggs at the 2-cell stage was calculated; 48 h after the beginning of cultivation, the number of eggs at each of the 3-cell stage, 4-cell stage, and 8-cell stage was calculated; 72 h after the beginning of cultivation, the numbers of morulas and blastocysts were calculated; and 96 h after the beginning of cultivation, the number of blastocysts was calculated. The number of embryos at each stage was recorded in Table 11, and the development rate (the number of fertilized eggs at 0 h was counted as 100%) was shown in Table 12. For the evaluation of development rate, with the number of fertilized eggs (at 0 h) as 100%, and a proportion of embryos developing normally was calculated at 24 h, 48 h, 72 h, and 96 h, which was the normal development rate. In addition, due to natural mating, the recovered fertilized eggs (at 0 h) included some unfertilized eggs, and even when a fertilized egg colony (at 0 h) recovered in the same experiment was allocated to fertilized egg colonies (at 0 h) under different conditions, the unfertilized egg content may also change occasionally. A development rate was also calculated with the number of embryos at the 2-cell stage 24 h after fertilization as 100%, and the calculation was conducted to exclude the influence of the unfertilized egg content that accidentally changed among condition groups. That is, in order to calculate the development rate based on the number of eggs that had actually started embryonic development, the number of eggs at the 2-cell stage 24 h after fertilization was set to 100%, and the development rate in this case was shown in Table 13. The statistical analysis of each result was conducted by chi-square test, and there were statistical significant differences at p<0.05 (*) and p<0.01 (**). Since the control exosome relatively reduced the embryonic development, statistical comparison was conducted relative to the medium group without any exosome.

TABLE 11Number of developing embryos in each group024487296GrouphhhhhOnly medium301818102Control exosome30171760CD56brightCD16−CD49a+-positive dNK3023211515cell-derived exosomeCD56brightCD16−CD49a+CD39+-positive3023201514dNK cell subset-derived exosomeCD56brightCD16−CD49a+CD27+-positive3024201514dNK cell subset-derived exosomeCD56brightCD16−CD49a+CD160+-positive3024211514dNK cell subset-derived exosomeCD56brightCD16−CD49a+TIGIT+-positive3024211515dNK cell subset-derived exosomeCD56brightCD16−CD49a+CD39+TIGIT+-3023211515positive dNK cell subset-derived exosome

TABLE 12Development rate of each group (%) calculated withthe number of fertilized eggs at 0 h as 100%024487296GrouphhhhhOnly medium100.0060.0060.0033.336.67Control exosome100.0056.6756.6720.000.00*CD56brightCD16−CD49a+-positive dNK cell-derived100.0076.6770.0050.00*50.00**exosomeCD56brightCD16−CD49a+CD39+-positive dNK cell100.0076.6766.6750.00*46.67**subset-derived exosomeCD56brightCD16−CD49a+CD27+-positive dNK cell100.0080.0066.6750.00*46.67**subset-derived exosomeCD56brightCD16−CD49a+CD160+-positive dNK cell100.0080.0070.0050.00*46.67**subset-derived exosomeCD56brightCD16−CD49a+TIGIT+-positive dNK cell100.0080.0070.0050.00*50.00**subset-derived exosomeCD56brightCD 16−CD49a+CD39+TIGIT+-positive100.0076.6770.0050.00*50.00**dNK cell subset-derived exosome

TABLE 13Development rate of each group (%) calculated withthe number of fertilized eggs at 24 h as 100%Group24 h48 h72 h96 hOnly medium100.00100.0055.5611.11Control exosome100.00100.0035.290.00*CD56brightCD16−CD49a+-positive dNK cell-derived100.0091.3065.2265.22**exosomeCD56brightCD16−CD49a+CD39+-positive dNK cell subset-100.0086.9665.2260.87**derived exosomeCD56brightCD16−CD49a+CD27+-positive dNK cell subset-100.0083.3362.5058.33**derived exosomeCD56brightCD16−CD49a+CD160+-positive dNK cell subset-100.0087.5062.5058.33**derived exosomeCD56brightCD16−CD49a+TIGIT+-positive dNK cell subset-100.0087.5062.5062.50**derived exosomeCD56brightCD16−CD49a+CD39+TIGIT+-positive dNK cell100.0091.3065.2265.22**subset-derived exosome

(chi-square test vs only medium group, **: p<0.01, and *: p<0.05)

Results showed that, when the number of fertilized eggs at 0 h was counted as 100%, compared with the control without any exosome, each group treated with an exosome derived from dNK cells or a dNK cell subset showed an increased development rate. The control exosome may have some cytotoxicity because it was derived from peripheral blood NK cells.

Example 12

Effect of the Exosomes on In Vitro Fertilized Eggs

5 U (units) of eCG was administered intraperitoneally to C57BL/6J female mice (3.9 to 4.0 weeks old), and 45 h to 48 h later, 5 U (units) of HCG (produced by ASKA Pharmaceutical Co., Ltd.) was administered intraperitoneally to induce excessive ovulation. 15 h after the administration of HCG, the laparotomy was conducted to collect fallopian tubes. In mineral oil, enlarged parts of the fallopian tubes were cut with a dissecting needle, and eggs were recovered into drops of mHTF medium. Sperm were recovered from the epididymal tail of C57BL/6J male mice, and then cultivated in mHTF medium at 37° C. and 5% CO2for 40 min to 1 h to achieve sperm capacitation. 2 μl to 4 μl of the sperm-containing mHTF medium was added to the medium drops with the collected eggs.

Fertilization was conducted at 37° C. and 5% CO2. 4 h to 6 h after fertilization, fertilized eggs were washed with KSOM medium to remove cumulus cells and sperm. The fertilized eggs were temporarily cultivated in a 37° C. and 5% CO2incubator with KSOM or mWM medium until all fertilized eggs were recovered.

Drops were formed with 100 μl of medium (which were overlaid with mineral oil (produced by Sigma-Aldrich), and some were added with the exosomes derived from dNK cells and dNK cell subsets and the control exosome described in Example 3 at a final concentration of 0.02%. 25 fertilized eggs were transferred into each drop, with 200 fertilized eggs for each treatment group. Then the fertilized eggs were cultivated. The number of embryos at the 2-cell stage 24 h after egg recovery and the number of blastocysts 96 h after egg recovery were determined. In addition, since the development of fertilized eggs obtained from in vitro fertilization would be slightly delayed, the number of blastocysts 120 h after the egg recovery was also determined. The number of embryos at the 2-cell stage 24 h after fertilization was counted as 100%, and on this basis, the embryo development rate at each cultivation time was calculated. Results were shown in Table 14.

TABLE 14Number of developing embryos in each groupGroup0 h24 h48 h72 h96 h120 hOnly medium200144101999190Control exosome200135111806055CD56brightCD16−CD49a+-positive200156144143140139dNK cell-derived exosomeCD56brightCD16−CD49a+CD39+-positive200150138135130129dNK cell subset-derived exosomeCD56brightCD16−CD49a+CD27+-positive200145131129125125dNK cell subset-derived exosomeCD56brightCD16−CD49a+CD160+-positive200140135133130129dNK cell subset-derived exosomeCD56brightCD16−CD49a+TIGIT+-positive200138125120120120dNK cell subset-derived exosomeCD56brightCD16−CD49a+CD39+TIGIT+-positive200141130122121121dNK cell subset-derived exosome

TABLE 15Development rate of each group (%) calculatedwith the number of fertilized eggs at 24 h as 100%Group24 h48 h72 h96 h120 hOnly medium100.0072.0050.5049.5045.50Control exosome100.0067.5055.5040.0030.00*CD56brightCD16−CD49a+-positive100.0078.0072.0071.5070.00*dNK cell-derived exosomeCD56brightCD16−CD49a+CD39+-positive100.0075.0069.0067.5065.00*dNK cell subset-derived exosomeCD56brightCD16−CD49a+CD27+-positive100.0072.5065.5064.5062.50*dNK cell subset-derived exosomeCD56brightCD16−CD49a+CD160+-positive100.0070.0067.5066.5065.00*dNK cell subset-derived exosomeCD56brightCD16−CD49a+TIGIT+-positive100.0069.0062.5060.0060.00*dNK cell subset-derived exosomeCD56brightCD16−CD49a+CD39+TIGIT+-positive100.0070.5065.0061.0060.50*dNK cell subset-derived exosome

(chi-square test vs only medium group, **: p<0.01, and *: p<0.05)

Results showed that, when the number of fertilized eggs at 24 h was counted as 100%, compared with the control without any exosome, each group treated with a dNK cell-derived exosome showed an increased development rate. The control exosome may have some cytotoxicity because it was derived from peripheral blood NK cells.

Example 13

Effect of the Exosomes on In Vitro Fertilized Egg Transfer

Embryos at the blastocyst stage in each group of Example 12 were transferred. Recipient mice were C57BL/6J female mice at 6 to 10 weeks old. The female mice were mated with vasectomized C57BL/6J male mice on the day before egg transfer, and on the next day, individuals with a vaginal plug were confirmed. General anesthesia was conducted with somnopentyl, and the back was incised to expose the uterus. The uterus was fixed with forceps, a 30 G injection needle was used to open a hole at the oviduct junction, and a glass capillary adsorbing a blastocyst was inserted to transfer the embryo into the uterus. After the transfer, the uterus was carefully put back into the body, and the retroperitoneum and skin were sutured. The blastocysts obtained from mice in each group were transferred into a corresponding recipient mice, separately. With the second day after egg collection being set as day 1, the caesarean section was conducted on day 19. The recipient mice were euthanized and laparotomized, and the uteruses were collected and the fetuses were taken out. An implantation rate was calculated according to the following formula: the number of implantation marks/the number of embryo transfers (Table 16), and a birth rate was calculated according to the following formula: the number of fetuses/the number of embryo transfers (Table 17).

TABLE 16Implantation rate of each groupNumberNumberofofimplan-embryoImplan-tationtrans-tationGroupmarksfersrate (%)Only medium429046.67Control exosome65510.91CD56brightCD16−CD49a+-positive dNK12313988.49*cell-derived exosomeCD56brightCD16−CD49a+CD39+-positive12112993.80**dNK cell subset-derived exosomeCD56brightCD16−CD49a+CD27+-positive11512592.00*dNK cell subset-derived exosomeCD56brightCD16−CD49a+CD160+-positive11012985.27*dNK cell subset-derived exosomeCD56brightCD16−CD49a+TIGIT+-positive9812081.67*dNK cell subset-derived exosomeCD56brigbtCD16−CD49a+CD39+TIGIT+-10512186.78*positive dNK cell subset-derived exosome

(chi-square test vs only medium group, **: p<0.01, and *: p<0.05)

TABLE 17Birth rate of each groupNumberNumberofBirthofembryorateGroupfetusestransfers(%)Only medium409044.44Control exosome2553.64CD56brightCD16−CD49a+-positive dNK12213987.77*cell-derived exosomeCD56brightCD16−CD49a+CD39+-positive11912992.25**dNK cell subset-derived exosomeCD56brightCD16−CD49a+CD27+-positive11012588.00*dNK cell subset-derived exosomeCD56brightCD16−CD49a+CD160+-positive9712975.19*dNK cell subset-derived exosomeCD56brightCD16−CD49a+TIGIT+-positive9212076.67*dNK cell subset-derived exosomeCD56brightCD16−CD49a+CD39+TIGIT+-10112183.47*positive dNK cell subset-derived exosome

(chi-square test vs only medium group, **: p<0.01, and *: p<0.05)

Results showed that, compared with the control without any exosome, each group treated with a dNK cell-derived exosome showed an increased implantation rate and an increased birth rate. The control exosome may have some cytotoxicity because it was derived from peripheral blood NK cells.

The basic principles, main features, and advantages of the present disclosure are shown and described above. It should be understood by those skilled in the art that, the present disclosure is not limited by the above examples, and the above examples and the description only illustrate the principle of the present disclosure. Various changes and modifications may be made to the present disclosure without departing from the spirit and scope of the present disclosure, and such changes and modifications all fall within the claimed scope of the present disclosure. The protection scope of the present disclosure is defined by the appended claims and equivalents thereof.