Materials and reagents
Neuro-2a cells (SCSP-5035) and U-87 MG cells (TCHu138) were purchased from the cell bank of the Typical Culture Preservation Committee, Chinese Academy of Sciences (CAS). Human 293T and Huh7 cells were kindly provided by Dr. Gao Liu (Department of Liver Surgery, Zhongshan Hospital, Shanghai Medical College, Fudan University). Dulbecco’s modified eagle medium (DMEM)/F-12 (Cat. No. 11330032), fetal bovine serum (FBS) (Cat. No. 10099141C), N-2 (Cat. No. 17502048), B-27 (Cat. No. 17504044), L-glutamine (Cat. No. 25030081), penicillin-streptomycin (P/S) (Cat. No. 15140122) were purchased from Gibco (Thermo Fisher Scientific, USA). Hoechst 33342 (Cat. No. H3570, Thermo Fisher Scientific, USA) was used to stain the cell nuclei. Epidermal growth factor (EGF) (Cat. No. AF-100-15) and basic fibroblast growth factor (bFGF) (Cat. No. AF-100-18B) were purchased from PeproTech (Cranbury, NJ, USA). Alexa Fluor® 647 mouse anti-nestin (Cat. No. 560393, 1:1000) and Alexa Fluor® 488 mouse anti-Sox2 (Cat. No. 560301) antibodies were from purchased BD Biosciences (NJ, USA). A mitochondrial membrane potential assay kit with JC-1 (Cat. No. C2006) and DAPI (Cat. No. C1005) was obtained from Beyotime (Shanghai, China). 2,3,5-Triphenyl tetrazolium chloride (TTC) was purchased from Adamas (Cat. No. 01057376, Shanghai, China). Silicon-coated monofilament sutures for rat tMCAO (Cat. No. L3800/L3600) were purchased from Guangzhou Jialing Biotechnology (Guangdong, China).
Neonatal C57BL/6 mice were purchased from Shanghai Lingchang Biotechnology Co., Ltd. (Shanghai, China). Adult male Sprague-Dawley rats (7-8 weeks old, 250-300 g) were obtained from Shanghai Super-B&K Laboratory Animal Corp. Ltd. (Shanghai, China). All experimental procedures and animal care were approved by the Animal Welfare and Ethics Group, Laboratory Animal Science Department, Fudan University and were carried out according to the Guidelines for the Care and Use of Laboratory Animals by the National Institutes of Health. All rats were housed in a controlled environment with appropriate light (light/darkness: 12 h), temperature (18-26 ℃) and humidity (50-70 %) conditions. All animals were provided a standard diet and water during the study. Experimental rats were grouped using the random number table method.
After being successfully isolated from the brains of neonatal mice (day 1), mNSCs were cultured in FBS-free DMEM/F-12 medium supplemented with 2 % (v/v) B27, 1 % (v/v) N-2, EGF (20 ng/mL), recombinant human bFGF (20 ng/mL), L-glutamine (2 mM) and P/S (P: 100 U/mL; S: 100 mg/mL). For adherent cell cultivation, mNSCs were cultured in plates, dishes or flasks precoated with Matrigel (Cat. No. 354277, Corning, NY, USA). The mNSCs were passaged and used upon reaching 80-90 % confluency. The mouse Neuro-2a cells, SV40T antigen-expressing human 293T cells, U-87 MG cells and human Huh7 cells were cultured in high-glucose DMEM supplemented with 10 % (v/v) heat-inactivated FBS and P/S (P: 100 U/mL; S: 100 mg/mL). After reaching 70-90 % confluency, the cultured cells were passaged and used. All cells were cultured in an incubator at 37 °C under an atmosphere with 5 % CO2.
Briefly, the medium of cultured cells was discarded, and the cells were washed with phosphate buffer saline (PBS) 3 times. Then, the cells were fixed with fixation buffer (Biolegend) for 20 min, washed with PBS, and then incubated with membrane-rupturing reagents for another 20 min. Subsequently, after being washed with PBS, the prepared cells were coincubated with specific primary antibodies for 1 h. Then, after being counterstained with 4’,6-diamidino-2-phenylindole (DAPI) for approximately 10 min, cell samples were observed and imaged with a confocal microscope (Nikon) or directly detected by flow cytometry analysis without nuclear staining. To assess mitochondrial colocalization with lysosomes, mitochondria from 293T cells expressing COX8A N-terminal signal peptide-mCherry fusion protein were isolated and incubated with a culture of normal 293T cells for 24 h, after which the cells were washed twice with PBS. Next, the cells were incubated with Lyso Dye (Cat. No. MD01, Dojindo Laboratories, Kumamoto, Japan) at 37℃ for 30 min. Then, after being counterstained with Hoechst 33342 at 37 ℃ for 10 min, the cells were observed and imaged with a confocal microscope (Nikon). All experimental procedures were conducted in darkness at room temperature (RT).
Transmission electron microscopy (TEM)
To observe the morphological characteristics of Neuro-2a cells and mNSCs, TEM was performed as described in previous studies[31, 32]. Briefly, cultured Neuro-2a cells and mNSCs were fixed with 2.5 % glutaraldehyde (2 h, RT) and then centrifuged (300 × g, 5 min). Subsequently, the harvested cells were postfixed with precooled 1 % osmic acid (2 h, 4 °C) and then centrifuged again (300 × g, 5 min). After gradient alcohol dehydration and penetration with a solution of acetone and epoxy resin at different proportions, the cell samples further embedded into epoxy resin and solidified for 48 h. Subsequently, the embedded samples were sectioned (thickness: 60-100 nm) and then double-stained with 3 % uranyl acetate and lead citrate. Finally, the stained sections were observed and imaged by TEM (Tecnai G2 20 TWIN，FEI Company, Oregon, USA).
The mitochondrial fluorescent dyes MitoTracker™ Red CMXRos (Cat. No. M7512, Thermo Fisher Scientific, Waltham, MA, USA), MitoBright Deep Red (Cat. No. MT12, Dojindo Laboratories, Kumamoto, Japan) and MitoTracker™ Green FM (Cat. No. M7514, Thermo Fisher Scientific, Waltham, MA, USA) were used to mark Neuro-2a cells or mNSCs, respectively, by coincubation at 37 °C for 45 min in darkness, after which the cells were washed 3 times with PBS and the supernatants were discarded. Next, the cells were counterstained with Hoechst 33342 (Cat. No. H3570, Thermo Fisher Scientific, Waltham, MA, USA) for 10 min, washed 3 times with PBS and then observed and imaged with a confocal microscope (Nikon). In addition, COX8A gene N-terminal signal peptide-mCherry fusion protein-expressing pLV-Mito-mCherry lentiviral vector (Cat. No. VL3512, Inovogen Tech. Co., Ltd., Chongqing, China) was used to transfect target cells (293T cells, MOI: 30) and mark mitochondria within cells. Subsequently, marked mitochondria were isolated from labeled or transfected cells for further experiments.
Isolation of mitochondria
Appropriate mitochondria from mNSCs and Neuro-2a cells were isolated using a Mitochondria Isolation kit for Cultured Cells (Cat. No. #89874, Thermo Fisher Scientific, USA) as previously described[32, 40] with minor modifications. Briefly, after the cultured cells were digested (trypsin) and centrifuged (300 × g, 5 min), the supernatant was removed and the cells were resuspended in mitochondria isolation reagent A (800 μL) in a 2.0-mL microcentrifuge tube before being vortexed for 5 s and then incubated for 2 min on ice. Then, reagent B (10 μL) was added and the sample was incubated in situ for 5 min. The sample was then vortexed at maximum speed 5 times (1 min each time) and then mixed with reagent C (800 μL). Subsequently, the mixture was centrifuged (700 × g, 10 min, 4 ℃), and the resulting supernatant was further centrifuged (12000 × g, 15 min, 4 ℃). Finally, fresh mitochondria were obtained and used for subsequent experiments.
Mitochondrial membrane potential (MMP) analysis
The MMP of cultured cells was assessed using a Mitochondrial Membrane Potential Assay kit with JC-1 (Cat. No. C2006, Beyotime Biotechnology, Shanghai, China) based on a previously described method[41, 42]. Single-cell suspensions of mNSCs and Neuro-2a cells were prepared and then coincubated with JC-1 working solution for 20 min at 37 ℃. Subsequently, sample cells were centrifuged (600 × g, 4 ℃, 5 min) and then washed with JC-1 buffer solution 2 times before being resuspended and assayed by flow cytometry.
Polymerase chain reaction (PCR)
Absolute quantitative PCR was performed as previously described with some modifications[43-45]. Briefly, genomic DNA (gDNA) from mNSCs and Neuro-2a cells was extracted using a HiPure Blood DNA Mini kit (Cat. No. D3111-03, Guangzhou Magen Biotechnology Co. Ltd., Guangdong, China) according to the manufacturer’s directions. The genes mt-ND1 and mt-RNR1 were used to assess mitochondrial DNA (mtDNA) levels, while the genes β-globin and β-actin were used to evaluate the nuclear DNA content. The sequences of the primers used to analyze these genes are described in detail in Table 1S. Then, the obtained gDNA was PCR amplified, detected by agarose gel electrophoresis and used to perform TA cloning. Next, plasmids were extracted from the positive colonies, and appropriate standards for absolute quantitation through quantitative PCR analysis were obtained. Then, the acquired standards were serially diluted to generate a standard curve. All DNA samples were analyzed by quantitative PCR using AceQ qPCR SYBR Green Master Mix (Cat. No. Q111-03, Vazyme Biotech Co., Ltd., Jiangsu, China) in a Real-time PCR Instrument (Applied Biosystems, Thermo Fisher Scientific, Waltham, MA, USA). Finally, the copy numbers of mt-ND1, mt-RNR1, β-globin and β-actin were determined with the calculated CT values and standard curves. Finally, the relative abundance of mitochondria was reported as the ratio of mtDNA to nuclear DNA (mt-ND1/β-globin, mt-RNR1/β-actin). The experiment was repeated 3 times.
Mitochondrial stress test of cultured cells
Mitochondrial stress tests were performed using the Seahorse XF analysis platform according to previous methods[33, 45, 46]. Briefly, Neuro-2a cells and mNSCs were seeded onto 96-well XF-96 plates (Seahorse Biosciences, Billerica, MA, USA) precoated with Matrigel. The oxygen consumption rate (OCR) values of cultured cells were measured using a Seahorse XFe-96 Extracellular Flux Analyzer (Seahorse Biosciences, Billerica, MA, USA) for untreated cells (basic OCR) or following the addition of mitochondrial respiration inhibitors to the system. The ATP synthase inhibitor oligomycin (10 μM), the oxidative phosphorylation uncoupler carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP; 10 μM) and the electron transport chain inhibitor rotenone/antimycin (5 μM) were added to the system to assess mitochondrial oxidative respiration activity through OCR measurements. The obtained OCR values were normalized for total protein content per well.
Metabolomic analysis was performed using the liquid chromatography-mass spectrometry (LC-MS) method as described in previous reports [47, 48]. mNSCs and Neuro-2a cells (n = 8) were seeded onto the petri dishes (10 cm) and cultured for 24 h, harvested upon reaching 90 % confluency and then resuspended in 1 mL of a precooled chromatographic grade methanol-acetonitrile-water solution. Then, the samples were vortexed for 1 min, lysed by ultrasonication (30 min, 2 times) in an ice water bath and then incubated for 1 h at -20 ℃. After being centrifuged (14000 × g, 4 ℃, 20 min), the obtained cell samples were stored at -80 ℃ for subsequent use. Then, hydrophilic interaction liquid chromatography (HILIC) was used for LC separation of the samples with an Agilent 1290 Infinity LC ultra-performance liquid chromatography (UPLC) system (25℃, 0.3 mL/min). Then, the obtained samples were further analyzed by MS in the electrospray ionization source (ESI)-based cationic and anionic modes using a Triple TOF 5600 Mass Spectrometer (AB SCIEX, USA). Subsequently, the acquired raw LC-MS/MS data were converted to .mzXML format using ProteoWizard (ProteoWizard, Palo Alto, CA, USA) and then processed and analyzed with the XCMS package in the R software environment for peak alignment, retention time correction and peak area extraction. Next, the structures of metabolites were identified by an exact mass number matching (< 25 ppm) and secondary spectrum matching based on a self-built database. Last, the R software environment (R Foundation for Statistical Computing, Vienna, Austria) and MetaboAnalyst 4.0 online tools (http://www.metaboanalyst.ca) were used to perform principal component analysis (PCA), cluster analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis.
To assess the tumorigenicity of grafted mitochondria in vivo, mitochondria isolated from Neuro-2a cells and mNSCs were separately injected into the right infra-axillary of nude mice. Macroscopic images were acquired with a digital camera at 6 months after cellular or mitochondrial injection. Different types of mouse tissues (heart, liver, spleen, lung, kidney and injection site-related subcutaneous lymph node tissues) were collected after 6 months and prepared for perform hematoxylin & eosin (H&E) staining after the transplantation of mitochondria from Neuro-2a cells or mNSCs. Briefly, prepared paraffin-embedded tissue sections were stained with eosin for 10 s and then counterstained with hematoxylin for 5 min. Following dehydration in a graded ethanol series and being cleared in xylene, the sections were mounted with neutral balsam, and images were obtained with a microscope (Leica, DM2500, Germany).
Hypoxia-reoxygenation (H/R) analysis
H/R injury of Neuro-2a cells was performed according to a previously described method with minor modifications. Briefly, experimental cells were placed in a hypoxic environment (1 % O2, 5 % CO2 and 94 % N2) generated using a three gas incubator for 48 h and then further cultivated under conventional conditions (reoxygenation) for 24 h. The cultured cells were divided into 3 groups: control group [conventional culture (48 h) + replacing medium + continuing conventional culture (24 h)], H/R group [hypoxic culture (48 h) + replacing medium + continuing conventional culture (24 h)] and H/R + mitochondrial treatment group [hypoxic culture (48 h) + replacing medium (containing exogenous mitochondria) + continuing conventional culture (24 h)].
The viability of Neuro-2a cells was using a Cell Counting kit-8 (CCK-8) (Cat. No. CK04, Dojindo Laboratories, Kumamoto, Japan) as previously described[50-52]. Briefly, cultured Neuro-2a cells were incubated with CCK-8 working solution at 37 ℃ for 3 h in darkness. Then, the CCK-8 working solution-treated Neuro-2a cells were analyzed at 450 nm using a multifunctional microplate reader (Molecular Devices, Sunnyvale, CA, USA) to obtain optical density values.
Detection of ROS levels
DCFH-DA probes (Cat. No. S0033M, Beyotime Biotechnology, Shanghai, China) were used to measure the ROS levels in Neuro-2a cells according to the manufacturer’s instructions[53, 54]. Briefly, after being incubated with DCFH-DA probes (10 μmol/L, excitation and emission wavelengths of 488 and 525 nm, respectively) at 37 ℃ for 30 min, Neuro-2a cells were analyzed using a multifunctional microplate reader (Molecular Devices, Sunnyvale, CA, USA) or collected by centrifugation (300 × g, 5 min). Then, after being resuspended in PBS, the DCFH-DA-labeled Neuro-2a cells were further analyzed by flow cytometry.
Western blot (WB) analysis
WB analysis was performed as previously described[53, 55]. The following primary antibodies were used for WB detection: anti-MFN1 (Cat. No. 13798-1-AP, 1:500), anti-OPA1 (Cat. No. 27733-1-AP, 1:1000) and anti-DRP1 antibodies (Cat. No. 12957-1-AP, 1:1000) were all purchased from Proteintech (Chicago, IL, USA); and anti-Bax (Cat. No. ab182733, 1:2000), anti-Bcl-2 (Cat. No. ab182858, 1:2000), anti-caspase-3 (Cat. No. ab184787, 1:2000) and anti-GAPDH antibodies (Cat. No. ab181602, 1:10000) were all purchased from Abcam (Cambridge, Cambs, UK). The anti-GAPDH antibody was used as an internal reference. Protein expression levels were detected using a Gel-Pro Analyzer (Media Cybernetics, MD, USA).
Detection of apoptosis levels
Neuro-2a cell apoptosis was evaluated using an Annexin V-FITC/PI Apoptosis Detection kit (Cat. No. 556547, BD Biosciences, Franklin Lakes, NJ, USA) as previously described[56, 57]. Briefly, prepared Neuro-2a cells were co-cultured with Annexin V-FITC dyes, after which propidium iodide was used to counterstain the treated cells. After being further cultivated for 15 min at RT in darkness, the double-stained Neuro-2a cells were detected by flow cytometry.
Transcriptomic detection and analysis of cultured Neuro-2a cells was performed as previously described with some modifications[58, 59]. Total RNA was extracted from cultured Neuro-2a cells with TRIzol reagent (Cat. No. 15596018, Thermo Fisher Scientific, USA) and then dissolved into DNase/RNase-free water (Cat. No. ST876, Beyotime Biotechnology, Shanghai, China). The purity and quantity of RNA samples were evaluated using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and an Agilent Bioanalyzer 2100 system (Agilent Technologies, Palo Alto, CA, USA). Then, the RNA samples were used for transcriptomic analysis. Subsequently, the acquired data were used for downstream analyses, including PCA and KEGG pathway enrichment analysis using the R software environment (R Foundation for Statistical Computing, Vienna, Austria). Subsequently, the data were visualized with a PCA plot, a heatmap, a Venn diagram and a bubble chart.
Rat tMCAO and mitochondrial delivery
Adult male Sprague-Dawley rats were used to induce tMCAO injury according to a previously described method with some modifications[60, 61]. In brief, after being anesthetized by an intraperitoneal injection of 2 % pentobarbital sodium (45 mg/kg), the rats were placed in a prone position. Then, after the left common carotid artery, external carotid artery (ECA) and internal carotid artery (ICA) were exposed, a silicon-coated monofilament suture was gradually inserted through the left ECA and was moved up into the left ICA to successfully occlude the left middle cerebral artery (MCA) and remained in situ for 120 min. Subsequently, the suture was carefully removed, the ECA was permanently ligated, and the incision was sutured. Sham-operated rats were subjected to the same procedure except that the 120 min-occlusion of the MCA with a silicon-coated monofilament suture was not performed. Experimental animals were then placed into individual cages and provided a standard diet and water. For intravital delivery of exogenous mitochondria, the prepared mitochondrial solution (isolated mitochondria, 10 μL) or vehicle solution (PBS, 10 μL) was immediately injected into the ICA after reperfusion started.
Evaluation of behavioral deficits
Neurobehavioral defects were evaluated 24 h after mitochondrial transplantation using multiple rating scales, including the Clark general functional deficit score[62, 63], the Clark focal functional deficit score[62, 63], the modified neurological severity score (mNSS)[61, 64] and the Rotarod test[61, 65] as previously described. Behavioral assessments were conducted by two skillful investigators who were both blinded to the animal groups.
TTC was used to evaluate the brain infarct size of tMCAO rats as previously described with minor modifications[66, 67]. Briefly, 24 h after tMCAO, the rats were deeply anesthetized and transcardially perfused with PBS to clear blood components of the brain vascular system, after which the rat brains were obtained and cut into 2-mm-thick coronal sections. Subsequently, the brain sections were incubated with a 2 % TTC solution at 37 °C for 30 min in darkness. Then, stained slices were placed from the frontal to occipital order, and macroscopic images were obtained with a digital camera. Infarct areas were calculated using Adobe Photoshop 21.0.0 (Adobe Systems Inc., San Jose, CA, USA).
Data that satisfied a Gaussian distribution (Shapiro-Wilk test) and homogeneity of variance (F-test) are presented as the means ± standard deviation (SD), and Student’s t-test or one-way analysis of variance (ANOVA) were used to compare the differences between two groups or among multiple groups, respectively. Data with a nonnormal distribution are presented as the medians (25 % and 75 % quantiles), and Mann-Whitney U-test was taken into consideration. Statistical analysis and diagram generation were performed using GraphPad Prism 8.0.1 (GraphPad Software, Inc., San Diego, CA, USA). A P-value < 0.05 was considered to indicate a significant difference.