Mitochondrial Transfer from Mouse Adipose-Derived Mesenchymal Stem Cells into Aged Mouse Oocytes

Summary

Here, we describe the isolation of mitochondria from mouse adipose-derived mesenchymal stem cells, and then transfer the mitochondria into aged mouse oocytes to improve the quality of the oocytes.

Abstract

Due to the decline in the quantity and quality of oocytes related to age, the fertility of women over 35 years of age has declined sharply. The molecular mechanisms that maintain oocyte quality remain unclear, thus it is difficult to increase the birth rate of women over 35 years old at present. Oocytes contain more mitochondria than any type of cell in the body, and any mitochondrial dysfunction can lead to reduced oocyte quality. In the 1990s, oocyte cytoplasmic transfer resulted in great success in human reproduction but was accompanied by ethical controversies. Autologous mitochondrial transplantation is expected to be a useful technique to increase the quality of oocytes that have decreased due to age. In the present study, we used adipose-derived stem cells from aged mice as a mitochondria donor to increase the quality of oocytes of aged mice. Further development of autologous mitochondrial transfer technology will provide a new and effective treatment for infertility in aged women.

Introduction

One of the important factors that affects female fertility is oocyte aging; decline in oocyte quality is the main cause of infertility in aged women. However, the main cause of oocyte aging and the molecular mechanism that regulates oocyte quality are still unclear. Previous studies have indicated that both the number and quality of mitochondria are involved in the quality control of oocytes and embryonic development^1,2,3. The decrease in the quantity and quality of mitochondria is closely related to aging³.

Many attempts have been made to improve the function of mitochondria in aged oocytes, including the nutritional supplement of mitochondria and mitochondria transfer. Well-known, effective, nutritional supplements of mitochondria include Coenzyme Q10 (CoQ10), Alpha-lipoic acid (α-LA), and resveratrol (RSV)⁴. Studies have shown that CoQ10 supplementation can not only improve the age-related decline in the quantity and quality of oocytes, but also promote the normal development and ovulation of oocytes⁵. α-LA slows the oocyte quality decline related to aging and the metabolic phenotype of patients with polycystic ovary syndrome (PCOS)^6,7. Resveratrol can reduce the number of oocytes with abnormal spindles and improper chromosome alignment increased in aging mice, while affecting the embryonic development in a dose-dependent manner⁸. However, as the clinical effect of nutritional supplements of mitochondria has not reached expected levels, other effective treatments need to be explored.

The first attempt of mitochondria transfer was carried out in 1997. The transfer of young donor oocyte cytoplasm into aged recipient oocytes improved the oocyte quality of the aged patients, who gave birth to healthy infants successfully⁹, which was the rationale behind the use of this technique. However, allogeneic oocyte cytoplasmic transfer cannot be applied to clinical practice due to two main reasons-the problem of genetic heterogeneity and regulatory issues caused by donor mitochondria transplantation. A previous study showed that autologous cell mitochondrial transplantation could improve the quality of oocytes, embryo development, and fertility of aged mice¹⁰, which had no ethical problems or genetic heterogeneity issues and solved some problems caused by the transfer of donor oocyte cytoplasm into recipient oocytes^10,11.

Meanwhile, autologous cell mitochondrial transfer was superior to the effect of previous nutritional supplements of mitochondria on improving the quality of oocytes¹¹. Therefore, autologous cell mitochondrial transplantation is the appropriate choice for the clinical application of this technology¹². Adipose-derived stem cells (ADSCs) can be obtained by minimally invasive technology, are easy to isolate and culture, and can be an ideal "seed" cell for regenerative medicine. Mitochondria are rich in ADSCs, and the function of mitochondria does not decline with age, which suggests that ADSCs are an excellent source of mitochondria^13,14. In this protocol, we introduce a method to transfer the mitochondria of mouse adipose-derived mesenchymal stem cells into aged mouse oocytes to improve the oocyte quality. This is a useful model for human ADSC autologous mitochondrial transfer technology.

Protocol

All the animal experiments described were approved by the Animal Research Ethics Committee of the Third Affiliated Hospital, Soochow University. All operations follow appropriate animal care and use agency and national guidelines. See the Table of Materials for details of all materials, instruments, and reagents used in this protocol.

1. Isolation and characterization of aged mouse adipose-derived mesenchymal stem cells (ADSCs)

1. Sacrifice the aged mice (10-month-old, an average of three) by cervical dislocation under pentobarbital sodium anesthesia and soak them in 75% alcohol for 5 min before adipose tissue isolation.
   NOTE: Soaking in 75% alcohol for 5 min can effectively reduce the contamination of primary cells.
2. Make a 2 cm incision in the skin on the bilateral inguinal, expose the subcutaneous fat, and use tweezers to isolate the subcutaneous fat. Collect the bilateral inguinal fat from the mice using sterilized ophthalmic scissors (avoid cutting the subcutaneous tissue). Place the adipose tissue in a 15 mL sterile centrifuge tube on ice and immediately transport it to the cell culture laboratory.
3. Transfer the adipose tissue to a 6-well plate, rinse it 3x with phosphate-buffered saline (PBS) containing 100 U/mL of penicillin and streptomycin. Remove the blood vessels, fatty fascia, and connective tissue under the adipose tissue and cut them into ~0.5 cm x 0.5 cm x 0.5 cm pieces.
   NOTE: When isolating adipose stem cells, there is often contamination of vascular endothelial cells. Removal of the blood vessels from adipose tissue can reduce vascular endothelial cell contamination.
4. Transfer the shredded adipose tissue to the same volume of collagenase type I solution (0.1% final concentration of collagenase type I) and digest at 37 °C for 30 min.
5. Add an equal volume of complete culture medium (DMEM F-12 medium containing 10% fetal bovine serum) to neutralize the collagenase type I, centrifuge at 600 × g for 10 min, and discard the supernatant and adipose tissue.
6. Resuspend the cell pellet in the complete culture medium. Remove the undigested tissue by filtration through a 40 µm cell strainer, and then centrifuge at 600 × g for 5 min.
7. Resuspend the cell suspension with 5 mL of the complete culture medium and use a 1 mL pipette to gently pipette up and down to form a single-cell suspension. Check the cell suspension under a microscope by placing an aliquot on a hemocytometer to confirm that there are only singe cells, without any aggregates. Add the single-cell suspension to a 25 cm² cell culture flask and culture in a 5% CO₂ incubator at 37 °C.
8. Change the culture medium 48 h later. Continue to change the culture medium every 2-3 days.
9. After 7-10 days, detach the cells for cell passaging when their confluency reaches 80%-90%. Remove the complete culture medium and wash the cells with 2 mL of PBS. Then, add 2 mL of 0.05% trypsin/EDTA to perform the cell dissociation^15,16.
10. Add adipogenic induction medium for 14 days to induce differentiation of the ADSCs into adipoctyes. Add osteogenic induction medium for 28 days to the ADSCs plated on 2% gelatin coated 24-well plates to induce their differentiation into osteoblasts. For neural differentiation, culture the ADSCs with neural induction medium, then detect neuron-specific enolase and neurofilament mediator polypeptide using immunofluorescence¹⁵.
    1. Fix the cells in 4% paraformaldehyde for 30 min at room temperature, followed by permeabilization with 0.5% Triton X-100 in PBS for 15 min.
    2. After blocking with 3% BSA in PBS, incubate the cells with primary antibodies (see Table of Materials) diluted in blocking solution at 4 °C overnight.
    3. Incubate the cells with secondary antibodies for 45 min and then stain them with DAPI for 10 min. Then, examine the cells with a fluorescence microscope.
11. Collect the single-cell ADSC suspension using 0.05% trypsin/EDTA, centrifuge at 600 × g for 5 min at room temperature, and resuspend in PBS to adjust the cell density to 1 × 10⁶/mL. Aliquot the cell suspension into 1.5 mL microcentrifuge tubes (100 µL/tube) and add FITC-labeled rabbit anti-mouse CD29, CD90, CD34, and HLA-DR monoclonal antibodies, with an antibody concentration of 0.5 µg/mL. Treat the control group with the same volume of rabbit IgG FITC, incubate on ice for 30 min, and rinse with PBS to remove the unconjugated antibody. Detect the ADSC surface markers by flow cytometry¹³.

2. Isolation of mitochondria from adipose stem cells

NOTE: All the mitochondria isolation operations must be carried out on ice.

1. When the ADSCs grow to 90% density, digest the cells with 2 mL of 0.05% trypsin/EDTA, centrifuge at 600 × g for 5 min, collect and count the cells, and take 1 × 10⁷ cells for each extraction.
2. Resuspend the cells in 2 mL of mitochondria extraction buffer (for buffer preparation: 10 mL of 0.1 M Tris-MOPS, 1 mL of 0.1 M EGTA/Tris, and 20 mL of 1 M sucrose; bring the volume to 100 mL with distilled water and adjust the pH to 7.4) with 1x protease inhibitor cocktail after washing with 2 mL of sterile PBS, and then incubate on ice for 5 min.
3. Homogenize the cells 20x-30x with a glass homogenizer (2.0 mL) and check the degree of homogenization by trypan blue staining.
   NOTE: The proportion of the stained cells should be ~80%. Excessive homogenization will damage the structure of the mitochondria.
4. Centrifuge the cell homogenate at 600 × g for 15 min, take the supernatant into a new, precooled 1.5 mL microcentrifuge tube, and repeat the operation once.
5. Centrifuge the supernatant at 7,500 × g for 15 min and discard the supernatant. Wash the mitochondrial precipitate and resuspend it by adding ~100 µL of precooled mitochondrial injection buffer (225 mM mannitol, 75 mM sucrose, 10 mM KCl, 10 mM Tris-HCl, and 5 mM KH₂PO₄, pH 7.2), at a concentration of 1-5 mg/mL (total protein concentration). Transport the mitochondrial suspension on ice.
6. Analyze the function and purity of the isolated mitochondria by JC-1 flow cytometric analysis and Western blot (VDAC1, β-actin, and Lamin B)¹³.
   1. Divide the isolated mitochondria into three groups: dimethyl sulfoxide (DMSO), JC-1 stained, and JC-1 stained with 30 min carbonyl cyanide 3-chlorophenylhydrazone (CCCP) pretreatment.
   2. After centrifuging at 7,500 × g, discard the supernatant and suspend the buffer.
   3. Capture the fluorescence intensity of the JC-1 monomers (FITC channel) and aggregates (PE channel) that were captured by flow cytometry. Evaluate the isolated mitochondrial membrane potentials by measuring the ratios of average fluorescence intensity of the PE channel to the FITC channel.
      ​NOTE: Perform the above steps in the dark.

3. Ovarian superstimulation

1. Inject 10-month-old female C57BL/6 mice intraperitoneally with 10 IU of pregnant mare serum gonadotropin (PMSG) for superovulation. After 48 h, inject 10 IU of human chorionic gonadotropin (hCG) intraperitoneally.
2. At 13 h after the injection of hCG, tear the swollen fallopian tube with microscopic tweezers. Release the cumulus complexes from the swollen fallopian tube. Dissolve the cumulus cells in M2 medium with hyaluronidase (0.3 mg/mL) at 37 °C.
   ​NOTE: The dissolution time is less than 5 min. Minimize the time for which the cumulus complex is exposed to hyaluronidase, as prolonged exposure can impair oocyte developmental potential.

4. Mitochondrial transfer along with ICSI

1. Put the sperm without a tail in the mitochondrial liquid. Obtain sperm without a tail by cutting using ultrasound¹⁷. Place the sperm without a tail in the mitochondrial liquid, such that there are 100 sperm in 50 µL of the mitochondrial liquid.
   NOTE: As the mitochondrial suspension is slightly viscous and not stable in vitro, complete the injection within 30 min and change the microinjection needle immediately when it becomes clogged. The optimal inner diameter of the microinjection needle is 5 µm.
2. Inject ~2 pL of mitochondrial respiration buffer or mitochondrial suspension with sperm into the oocytes using a microinjector under an inverted microscope (200x; see Table of Materials) within 30 min according to the protocol of Hiramoto as described by Mehlmann and Kline^10,18.
   NOTE: The injected mitochondrial suspension accounts for 1%-3% of the total volume of oocytes. Control the volume (1%-3%) of the mitochondrial suspension in the cytoplasm to ensure the consistency of the injection.
3. After injection, place the fertilized oocytes in M2 medium for equilibration for 15 min, and transfer the surviving fertilized eggs to M16 culture medium.
4. Examine the surviving fertile eggs by observing the smooth morphology, pronuclear formation, and release of the second polar body¹⁹. Observe, photograph, and count the embryos at 09:00 a.m. and 06:00 p.m. every day for 4 days. Finally, collect and freeze the blastocysts and store them in a -80 °C refrigerator for ATP and mtDNA copy number determination to evaluate the number and improvement of mitochondrial transplantation.
   1. Prepare standard plasmid (plasmid for absolute quantification, as previously described¹³), for mitochondrial copy number determination according to the copy number of 1 × 10⁷, 1 × 10⁶, 1 × 10⁵, 1 × 10⁴, 1 × 10³, 1 × 10², and 1 × 10¹. Transfer the embryos to the sterilization tube and add 20 µL of lysate buffer to release the mitochondrial DNA. Then, inactivate the protease in the lysate at 55 °C for 20 min and 95 °C for 10 min.
   2. Determine the mtDNA copy number by fluorescence quantitative PCR using the following setup: 5 µL of PCR mix, 0.5 µL of B6 primer-forward, 0.5 µL of B6 primer-rev, 2 µL of pyrolysis product, and 2 µL of ddH₂O. Follow the PCR conditions: stage 1, 95 °C for 3 min; stage2, 95 °C for 30 s, 58 °C for 30 s, and 72 °C for 30s; repeat the cycle 40x.

Results

In this protocol, we isolated and characterized ADSCs from mouse fat (Figure 1). To obtain isolated mitochondria, the cell membrane must be disrupted using a glass homogenizer. (Figure 2A). It is important to obtain a uniform mitochondrial fraction without large clumps so that the microinjection tube is not blocked. First, 200 µL, and then 10 µL, pipette tips must be used to resuspend the homogenates gently; finally, a 29 G needle must be used to slowl...

Discussion

Oocytes contain more mitochondria than any type of cell in the body, with ~1-5 × 10⁵ mtDNA copy numbers. Mitochondria are essential for oocyte maturation, fertilization, and embryonic development, thus, any mitochondrial dysfunction can lead to decreased oocyte quality. Decreased mitochondrial quantity and quality are closely related to physiological aging. In this protocol, a simple method for isolating mitochondria from the ADSCs of aged mice and transfer to aged mouse oocytes was introduced to attempt ...

Materials

Name	Company	Catalog Number	Comments
0.05% trypsin/EDTA	Gibco	25300054	Cell Culture
4% paraformaldehyde	beyotime	P0099	immunofluorescence
40 μm cell strainer	Corning	352340	ADSC isolation
adipogenic induction	Cyagen	HUXXC-90031	Multidirectional differentiation
Alizarin red staining solution	Sigma	A5533	Multidirectional differentiation
Antibody against CD29	BD Biosciences	558741	flow analysis
Antibody against CD34	BD Biosciences	560942	flow analysis
Antibody against CD90	BD Biosciences	553016	flow analysis
Antibody against HLA-DR	BD Biosciences	555560	flow analysis
β-actin	Abcam	ab-8226	Mitochondrial function test
BSA	Sigma	V900933	immunofluorescence
CCCP	Solarbio	C6700	mitochondria JC-1 flow analysis
ChamQ Universal SYBR qPCR Master Mix	Vazyme	Q711	qPCR
collagenase type I	Sigma	SCR103	ADSC isolation
DAPI	Invitrogen	D1306	immunofluorescence
DMEM-F12	Gibco	11320033	Cell Culture
DMSO	Sigma	276855	mitochondria JC-1 flow analysis
EGTA	Sigma	324626	Mitochondria isolation
FBS	Gibco	10100147	Cell Culture
Flow cytometry	BD Biosciences	FACSCanto™ II	Characteristics of ADSCs
fluorescence microscope	leica	DM2500	immunofluorescence
gelatin	Sigma	48722	Multidirectional differentiation
glass homogenization tube	Sangon	F519062	Mitochondria isolation
hCG	Aibei	M2520	Ovarian superstimulation
hyaluronidase	Sigma	H1115000	Ovarian superstimulation
Inverted microscope	Olympus	IMT-2	Microinjection
Isolated Mitochondria Staining Kit	Sigma	CS0760	mitochondria JC-1 flow analysis
JC-1	Sigma	T4069	Mitochondrial function test
KCl	Sigma	P5405	Mitochondria transfer
KH2PO4	Sigma	P5655	Mitochondria transfer
LaminB	Abcam	ab-16048	Mitochondrial function test
M16 Medium	Sigma	M7292	embryo cell culture
M2 Medium	Sigma	M7167	embryo cell culture
mannitol	Sigma	M9546	Mitochondria transfer
Microinjector	Olympus+ eppendorf	IX73	Mitochondria transfer
MitoTracker red	Invitrogen	M22425	Mitochondria staining
MOPS	Sigma	M1442	Mitochondria isolation
neurofilament mediator polypeptide (NFM)	Santa Cruz Biotechnology	sc-16143	Multidirectional differentiation
neurogenic induction	Gibco	A1647801	Multidirectional differentiation
Neuron-specific enolase (NSE)	Santa Cruz Biotechnology	sc-292097	Multidirectional differentiation
Oil Red O	Sangon	E607319	Adipogenic differentiation
oil red O solution	Sigma	O1516	Multidirectional differentiation
osteogenic induction	Cyagen	HUXXC-90021	Multidirectional differentiation
PBS (phosphate buffered saline)	Hyclone	SH30256.LS	Cell Culture
penicillin and streptomycin	Hyclone	SV30010	Cell Culture
PMSG	Aibei	M2620	Ovarian superstimulation
protease Inhibitor cocktail	Sigma	P8340	Mitochondria isolation
sucrose	Sigma	V900116	Mitochondria isolation
Tris	Sigma	648314	Mitochondria isolation
Tris-HCl	Sigma	108319	Mitochondria transfer
Triton X-100	beyotime	P0096	immunofluorescence
VDAC	Abcam	ab-14734	Mitochondrial function test