Urine-Derived Mesenchymal Stem Cell Mitochondria as Well as Transplantation Method and Use Thereof

Abstract

A urine-derived mesenchymal stem cell mitochondria as well as a transplantation method and use thereof. The urine-derived mesenchymal stem cell mitochondrion is extracted from urine to be used for improving the quality of oocytes. The transplantation method comprises: jointly injecting sperms and the urine-derived mesenchymal stem cell mitochondria into mature oocytes for blastaea culture during the intracytoplasmic sperm microinjection. The present disclosure has the beneficial effects: during the traditional ICSI in combination with the transplantation of the urine-derived mesenchymal stem cell mitochondria, the fertilization rate of human in-vitro fertilization and the quality of embryos are significantly improved; the urine-derived mesenchymal stem cell mitochondria of the present disclosure can be used for in-vitro fertilization of low-prognosis patients with infertility with a good treatment effect; the problem of an autologous mitochondrion source in the prior art is solved without involving the introduction of a third-party genetic material and ethical issues.

Claims

1. Urine-derived mesenchymal stem cell mitochondrion, wherein the urine-derived mesenchymal stem cell mitochondria is extracted by the following method: (1) collecting urine into a vessel, centrifuging, discarding supernatant, adding PBS buffer into the vessel for resuspension, centrifuging again, discarding the supernatant, resuspending cell precipitates using a urine-derived mesenchymal stem cell isolation culture medium, inoculating the resuspended cell precipitates into a gelatin-coated 6-well plate, and putting the 6-well plate into an incubator for primary culture; changing the culture medium once when cells clones are formed, digesting with pancreatin after the clones are fused into a blockbuster, sucking pancreatin after digestion, resuspending using a urine-derived mesenchymal stem cell amplification culture medium, and inoculating into a new 6-well plate, and marking a P1 generation; (2) putting the P1 generation obtained in step (1) in the incubator to continue culture, digesting using pancreatin when the P1 generation grows to cover the 85%-95% of area of the 6-well plate, sucking the pancreatin after digestion, resuspending using the urine-derived mesenchymal stem cell amplification culture medium, centrifuging, discarding the supernatant, adding a cell lysis solution, lysing on ice, then adding a mitochondrion extraction solution, and uniformly mixing; and centrifuging, sucking the supernatant, transferring into another vessel, centrifuging again, and discarding the supernatant to obtain precipitates namely mitochondria; and (3) centrifuging again, washing, discarding the supernatant, resuspending the mitochondrion precipitates using a mitochondrion preservation solution, and storing at 0-4° C.

2. The urine-derived mesenchymal stem cell mitochondria according to claim 1, wherein in step (1), the centrifuging is performed for 10 min at 120 rpm; the time for cell clone formation is 7 days, and the time of fusing the clones into blockbuster is 14 days.

3. The urine-derived mesenchymal stem cell mitochondria according to claim 1, wherein in step (1), the gelatin is a 0.1% gelatin aqueous solution; in step (1) and step (2), the incubators are all incubators with 37° C. and 5% CO.sub.2; the pancreatin is a 0.05% pancreatin aqueous solution; and the digestion time is 1 min.

4. The urine-derived mesenchymal stem cell mitochondria according to claim 1, wherein in step (2), the lysis time is 5 min; the redundant pancreatin is sucked after digestion using pancreatin, 1 mL of urine-derived mesenchymal stem cell amplification culture medium is added into each well for resuspension, the above resuspension is transferred into a centrifuge tube to be centrifuged for 3 min at 1200 rpm, the supernatant is discarded, 500 ul of cell lysis solution is added to lyze the cells on ice for 5 min, and 1 ml of mitochondrion extraction solution is then added and uniformly mixed.

5. The urine-derived mesenchymal stem cell mitochondria according to claim 1, wherein in step (2), 1 ml of mitochondrion extraction solution is added and uniformly mixed; the above mixture is centrifuged for 10 min at 800 g, the supernatant is sucked and transferred to another centrifuge tube, and then centrifuged for 10 min at 5000 g, and the supernatant is discarded to obtain precipitates namely mitochondria.

6. A transplantation method of the urine-derived mesenchymal stem cell mitochondria according to claim 1, the transplantation method comprising: during the intracytoplasmic sperm microinjection, sperms and urine-derived mesenchymal stem cell mitochondria are jointly injected into mature oocytes; preferably, the urine-derived mesenchymal stem cell mitochondria are autologous urine-derived mesenchyme cytoplasmic stem cell mitochondria.

7. The transplantation method of the urine-derived mesenchymal stem cell mitochondria according to claim 6, the transplantation method comprising: the sperms are grabbed and braked using a micromanipulation needle, the sperms are pressed against the tip of the injection needle and transferred into a urine-derived mesenchymal stem cell mitochondrion droplets and sucked for many times to form a homogenate, then, the urine-derived mesenchymal stem cell mitochondrion droplets together with the previously grabbed and braked sperms are injected into the cytoplasm of mature oocytes.

8. The transplantation method of the urine-derived mesenchymal stem cell mitochondrion according to claim 7, the transplantation method comprising: under a micromanipulation table, sperms are grabbed and braked using a micromanipulation needle with a diameter of 4.5 um, the sperms are pressed against the tip of the injection needle, transferred into mitochondrion droplets and sucked for many times to form a homogenate, the urine-derived mesenchymal stem cell mitochondrion droplets are sucked using the injection needle, and 40,000-60,000 mitochondria at the 10 um volume length of the front end of the injection needle together with sperms are injected into the cytoplasm of mature oocytes.

9. A drug for improving the quality of oocytes, the drug comprising the urine-derived mesenchymal stem cell mitochondria according to claim 1.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0044] To more clearly illustrate the embodiments of the present disclosure or technical solution in the prior art, drawings required to be used in the embodiments or in the prior art will be simply described below. Obviously, drawings used in the following description are only some embodiments of the present disclosure, and other drawings can also be made by persons of ordinary skill in the art without creative efforts according to these drawings.

[0045] FIG. 1 shows comparison of mitochondrion biological activity of mature oocytes from low-prognosis patients with those of normal people;

[0046] FIG. 2 is a light microscope image of P1-generation urinary-derived mesenchymal stem cells according to example 3 of the present disclosure;

[0047] FIG. 3 shows flow cytometric identification of urine-derived mesenchymal stem cells;

[0048] FIG. 4 is a diagram showing activity identification of urine-derived mesenchymal stem cell mitochondria extracted according to example 3 of the present disclosure;

[0049] FIG. 5 shows a process of joint injection of sperms and autologous urine-derived mesenchymal stem cell mitochondria during the ICSI according to the present disclosure;

[0050] FIG. 6 shows comparison of fertilization situations of control group and test group on the first day after in-vitro fertilization according to the present disclosure;

[0051] FIG. 7 shows comparison of embryo development situations of control group and test group on the third day after in-vitro fertilization according to the present disclosure;

[0052] FIG. 8 shows comparison of embryo development situations of control group and test group on the fifth day after in-vitro fertilization according to the present disclosure;

[0053] FIG. 9 shows comparison of mitochondrion copy numbers of urine-derived, bone-marrow and adipose-derived mesenchymal stem cells and ovarian granular cells;

[0054] FIG. 10 shows comparison of extracellular acid production capacities (ECAR) of urine-derived, bone-marrow and adipose-derived mesenchymal stem cells and ovarian granular cells;

[0055] FIG. 11 shows comparison of oxygen consumptions (OCR) of urine-derived, bone-marrow and adipose-derived mesenchymal stem cells and ovarian granular cells; and

[0056] FIG. 12 is an expression map of electron transport chain genes encoded by the mitochondria of urine-derived, bone marrow, adipose-derived mesenchymal stem cells and ovarian granular cells.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0057] To make the objective, technical solution and advantages of the present disclosure more clear, the technical solution of the present disclosure will be further described in detail through embodiments in combination with drawings. Obviously, the described embodiments are only a part of embodiments of the present disclosure but not all the embodiments. Based on the embodiments of the present disclosure, all other embodiments obtained by persons of ordinary skill in the art without creative efforts are included within the protective scope of the present disclosure.

[0058] The urine-derived mesenchymal stem cell isolation culture medium, the urine-derived mesenchymal stem cell amplification culture medium, the lysate, the mitochondrion extraction solution, pancreatin and a mitochondrion preservation solution used in the following examples are commercially available, in which the urine-derived mesenchymal stem cell isolation culture medium (Cat. No. AV-1501, Asia Vector); the urine-derived mesenchymal stem cell amplification culture medium (Cat. No. AV-1501-B, Asia Vector); the lysate (Cat. No. MITOISO2 component, Sigma); the mitochondrion extraction solution (product number MITOISO2 component, Sigma); the pancreatin (Sigma); the mitochondrion preservation solution (product number MITOISO2 component, Sigma).

Example 1

[0059] In this example, the urine-derived mesenchymal stem cell mitochondria were extracted by using the following method:

[0060] Urine was collected into a vessel and centrifuged, supernatant was discarded, PBS buffer was added into the vessel for resuspension, the resulting solution was centrifuged again, the supernatant was discarded, cell precipitates were resuspended using the urine-derived mesenchymal stem cell isolation culture medium, inoculated to a gelatin-coated 6-well plate, and the 6-well plate was put into an incubator for primary culture; the culture medium was completely changed once after cell clones were formed; after the clones are fused into a blockbuster, pancreatin was used for digestion, the pancreatin was sucked after digestion, the USC amplification culture medium was used for resuspension, the resuspended solution was inoculated into a new 6-well plate, and marked as a P1 generation (subsequent passage and culture were the same as those in this step).

[0061] (2) the P1 generation obtained in step (1) was placed in the incubator to continue culture, digestion was conducted using pancreatin when the P1 generation grown to cover the 95% of area of the 6-well plate, the pancreatin was sucked after digestion, resuspension was conducted using the urine-derived mesenchymal stem cell amplification culture medium, resuspended solution was centrifuged, the supernatant was discharded, a cell lysis solution was added for lysis on ice, then a mitochondrion extraction solution was then added, and uniformly mixed; and the mixed solution was centrifuged, the supernatant was sucked, transferred into another vessel and centrifuged again, and the supernatant was discarded to obtain precipitates namely mitochondria; and

[0062] (3) the obtained mitochondria were centrifuged again and washed, the supernatant was discarded, and the mitochondrion precipitate was resuspended using a mitochondrion preservation solution, and stored at the temperature of less than of 4° C.

Example 2

[0063] This example provides a transplantation method of urine-derived mesenchymal stem cell mitochondria, comprising: during the intracytoplasmic sperm microinjection, sperms and urine-derived mesenchymal stem cell mitochondria (mitochondrion solution droplets obtained by resuspending the mitochondrion precipitate using the mitochondrion preservation solution) were jointly injected into mature oocytes for blastula culture to improve the quality of oocytes, improve the fertility rate and embryo quality, and increase the embryonic development rate of human in-vitro fertilization.

Example 3

[0064] The Mitochondrion Biological Activity of Mature Oocytes of Low-Prognosis Patients in this Example was Compared with that of Normal People

[0065] The mitochondrion activities of mature oocytes in normal people and low-prognosis patients were observed under a confocal microscope. TMRE is tetramethylrhodamine ethyl ester, namely, a mitochondrion membrane potential indicator. Red fluorescence represents the intensity of mitochondrion activity. The results are shown in FIG. 1.

[0066] It can be seen from the results from FIG. 1 that the biological activity of oocyte mitochondria in normal people is significantly higher than that in low-prognosis patients.

[0067] The Urine-Derived Mesenchymal Stem Cell Mitochondria from the Same Low Prognosis Patient were Extracted Through the Following Steps (1)-(3):

[0068] 200 mL of urine from the above-mentioned low-prognosis patients was collected and dispensed into 50 mL sterile centrifuge tubes to be centrifuged at 1200 rpm for 10 min, supernatant was discarded, 20 mL of PBS was added for resuspension, the resulting resuspended solution was centrifuged again for 10 min at 1200 rpm, the supernatant was discarded, cell precipitates were resuspended using a USC isolation culture medium and then inoculated into a 0.1% gelatin-coated 6-well plate, the 6-well plate was placed in an incubator with 37° C. and 5% CO.sub.2 for primary culture. The culture medium was completely changed once after small clones were formed on the 7th day, and 0.05% pancreatin was added for digestion after the clones were fused into a blockblaster (filled with the entire 100×microscope field of view) on the 14th day, the redundant pancreatin was sucked after digestion for 1 min, the rest solution was resuspended using the USC amplification culture medium and then inoculated into a new 6-well plate, and then the inoculated product was marked as the P1 generation (the light microscope graph of P1-generation urinary-derived mesenchymal stem cells is shown in FIG. 2).

[0069] Flow Cytometry Surface Marker Identification of Urine-Derived Mesenchymal Stem Cells

[0070] Urine-derived mesenchymal stem cells detected by flow cytometry positively expressed mesenchymal stem cell surface positive markers CD29, CD73, CD90, CD13, CD44 and SSEA-4, negatively expressed hematopoietic stem cells and endothelial cells such as CD45, CD34, CD31 and HLA-DR, proving that their mesenchymal origins. The result is shown in FIG. 3. It can be seen from the figure that the isolated cells of the present disclosure are urine-derived mesenchymal stem cells.

[0071] (2) the P1 generation urine-derived mesenchymal stem cells obtained in step (1) were put in the incubator with 37° C. and 5% CO.sub.2 to continue culture, and the subsquent passage and culture are the same as them; pancreatin was used for digestion when the P1 generation grown to cover the 90% of area of the 6-well plate, the redundant pancreatin was sucked after digestion for 1 min, 1 mL of urine-derived mesenchymal stem cell amplification culture medium was used for resuspension, the supernatant was sucked to a 1.5 mL of sterile EP tube and centrifuged for 3 min at 1200 rpm, the supernatant was discarded, 500 ul of cell lysis solution was added for lysis on ice for 5 min (reversely and uniformly mixing every 1 min once), then 1 mL of mitochondrion extraction solution was added, and uniformly mixed; and the above mixed solution was centrifuged for 10 min at 5000 g, the supernatant was discarded to obtain precipitates namely mitochondria;

[0072] (3) the above mitochondria were centrifuged again at 5000 g for 10 min and washed, the supernatant was discarded, the precipitates were resuspended using 50 ul of mitochondrion preservation solution, and the resulting mitochondrion solution was stored on ice until use.

[0073] Urine-Derived Mesenchymal Stem Cell Mitochondria was Subjected to Activity Identification

[0074] After the urine-derived mesenchymal stem cell mitochondria were extracted, the mitochondrion activity was observed under the confocal microscope. The results are shown in FIG. 4. In the figure, TMRE is tetramethylrhodamine ethyl ester, that is to say, TMRE is used as the mitochondrion membrane potential indicator, red fluorescence represents the intensity of mitochondrion activity; FCCP is an oxidative phosphorylation uncoupling agent. The left picture shows that TMRE, rather than FCCP, is added in the urine-derived mesenchymal stem cell mitochondria obtained in example 3, the red fluorescence intensity shows that the urine-derived mesenchymal stem cell mitochondria have activity; the right picture shows that TMRE and FCCP are added as a negative control, after that, the red fluorescence is significantly weakened and the mitochondrion activity is significantly reduced.

[0075] Then, mature oocytes were used to perform ICSI in-vitro fertilization, specifically: under the micromanipulation table, sperms were grabbed and braked using a micromanipulation needle with a diameter of 4.5 um, the sperms were pressed against the tip of the injection needle, transferred into mitochondrion droplets and repeatedly sucked to suck the mitochondrion droplets (from mitochondrion droplets obtained by resuspending mitochondrion precipitates using 50 ul of mitochondrion preservation solution in step (3)) to ensure the mitochondrion concentration required for injection, m volume length of the front end together with the previously grabbed and braked sperms were injected into the cytoplasm of mature oocytes (shown in FIG. 5), the mitochondria were located on the tip of the injector, the sperms were behind the mitochondria, and then the cytoplasm was transferred into the embryo culture medium for blastocyst culture.

[0076] The traditional ICSI group (control group) from the same patient's sister eggs was compared with test group of autologous USC mitochondrion transplantation during ICSI.

[0077] Test group: ICSI mitochondrion co-injection in-vitro fertilization was performed on the sister eggs from 3 patients using the method in example 3;

[0078] Control group: the control group is different from the test group in that the control group used the traditional ICSI in-vitro fertilization without autologous urine-derived mesenchymal stem cell mitochondrion transplantation; the specific method was as follows: mature oocytes were used for the traditional ICSI in-vitro fertilization, under the micromanipulation table, sperms were grabbed and braked using a micromanipulation needle with a diameter of 4.5 um, the sperms were pressed against the tip of the injection needle and then injected into the cytoplasm of mature oocytes, and then the cytoplasm was transferred into the embryo culture medium for blastocyst culture after injection was completed.

[0079] The fertilization situations and embryo situations of control group and test group were compared, and the results are shown in FIGS. 6-8. It can be seen from the figures that on the first day, the control group was fertilized abnormally; the test group was fertilized normally with double pronucleus (2PN) formed (FIG. 6); on the third day, the control group had grade IV embryos (fragments) and the test group had 9 cells Grade III embryos have normal cleavage speed (FIG. 7); on the fifth day, embryo development in the control group was blocked, and the test group was BC-grade early blastocysts (FIG. 8). The above test shows that the normal fertilization rate, cleavage speed, and embryo quality of the test group are all improved.

[0080] The inventors comprehensively evaluated many autologous mesenchymal stem cells (bone marrow, fat and urine) from various levels in the aspects of mitochondrion function and metabolic capacity, and made safety verification. It was found that the urine-derived mesenchymal stem cell mitochondria are more similar to oocytes compared with other types of mesenchymal stem cells in terms of maturity, function and metabolic mode, specifically:

[0081] 1. The mitochondrion copy numbers of urine-derived (USC), bone-marrow (BMSC), adipose (ASC)-derived mesenchymal stem cells and ovarian granular cells (GC) were compared, and the results are shown in FIG. 9.

[0082] It can be seen from FIG. 9 that there are no significant difference in the mitochondrion copy numbers of GC, USC, BMSC and ASC among young people; the mitochondrion copy numbers of GC and BMSC of the elderly are significantly reduced compared with those of young people, while the mitochondrion copy numbers of USC and ASC are not significantly reduced; the mitochondrion copy number of the elderly USC is significantly higher than that of the elderly GC and BMSC.

[0083] 2. cell metabolism and mitochondrion functions of urine-derived (USC), bone marrow (BMSC), adipose (ASC)-derived mesenchymal stem cells and ovarian granular cells (GC) were compared, and the results are shown in FIG. 10 and FIG. 11. FIG. 10 shows extracellular acid production capability (ECAR) of the detected cells, and indirectly shows the cytosolic glycolysis capability; FIG. 11 shows the oxygen consumption (OCR) of the detected cells, which reflects the oxidative phosphorylation capacity of mitochondria.

[0084] It can be seen from FIG. 10 and FIG. 11 that the overall cell metabolism capability (including glycolysis and oxidative phosphorylation) of the urine-derived mesenchymal stem cells (USC) is higher than the overall cell metabolism capabilities of bone marrow, adipose-derived mesenchymal stem cells and ovarian granular cells.

[0085] 3. The expression patterns of the electron transport chain genes encoded by the mitochondria of urine-derived (USC), bone-marrow (BMSC), adipose (ASC)-derived mesenchymal stem cells and ovarian granulosa cells (GC) are shown in FIG. 12.

[0086] It can be seen from FIG. 12 that the expression level of the electron transport chain genes encoded by the urine-derived mesenchymal stem cell mitochondria (USC) is higher than that of other cell types in both young and old patients.

[0087] Through the above test, it can be seen that the urine-derived mesenchymal stem cell mitochondria have more application advantages than other types of mesenchymal stem cell mitochondria in terms of number, mitochondrion function, and gene expression pattern. In addition, the urine-derived mesenchymal stem cell mitochondria are suitably used as the first choice of the autologous mitochondrion source due to its characteristic of non-invasive and large-scale acquisition.

[0088] The above descriptions are only specific embodiments of the present disclosure, but the protective scope of the present disclosure is not limited thereto. Those skilled in the art can easily conceive that changes or substitutions should be included within the protective scope of the present disclosure. Therefore, the protective scope of the present disclosure shall be based on the protective scope of the claims.