Characterization of mitochondrial health from human peripheral blood mononuclear cells to cerebral organoids derived from induced pluripotent stem cells

Mitochondrial health plays a crucial role in human brain development and diseases. However, the evaluation of mitochondrial health in the brain is not incorporated into clinical practice due to ethical and logistical concerns. As a result, the development of targeted mitochondrial therapeutics remains a significant challenge due to the lack of appropriate patient-derived brain tissues. To address these unmet needs, we developed cerebral organoids (COs) from induced pluripotent stem cells (iPSCs) derived from human peripheral blood mononuclear cells (PBMCs) and monitored mitochondrial health from the primary, reprogrammed and differentiated stages. Our results show preserved mitochondrial genetics, function and treatment responses across PBMCs to iPSCs to COs, and measurable neuronal activity in the COs. We expect our approach will serve as a model for more widespread evaluation of mitochondrial health relevant to a wide range of human diseases using readily accessible patient peripheral (PBMCs) and stem-cell derived brain tissue samples.

. Schematic summary of the study design. Purple panel: An overview and a timeline of sample reprogramming and differentiation from peripheral blood mononuclear cells (PBMCs) to induced pluripotent stem cells (iPSCs) to cerebral organoids (COs) or H9 human embryonic stem cells (H9 hESCs) to COs. Red panel: An overview of electrophysiology experiments (action potentials, spontaneous activity, and sodium and potassium currents) in cerebral organoids. Blue panel: An overview of mitochondrial (mt-) genetics (mtDNA haplogroup, heteroplasmy and copy number), function (oxidative phosphorylation, ATP production and mitochondrial membrane potential) and morphology assessment across PBMCs to iPSCs to COs or H9 hESCs to COs. www.nature.com/scientificreports/ level, pluripotent markers in the iPSCs were similar to those observed in H9 hESCs ( Fig. 2B-v), further demonstrating successful generation of iPSCs from PBMCs. We previously established a robust protocol that allows for the reproducible production of COs from human pluripotent stem cells 17 . These COs display consistent cell type composition and proportions across different batches, making this CO platform useful for disease research as it addresses the problem of variability 17 . Using this protocol, we generated 4.5-month old COs from PBMCs-derived iPSCs (N = 14, Table S3) and demonstrated that they have similar overall morphology as those derived from the H9 hESCs (N = 14, Fig. 2C and Fig. S1-A). Immunofluorescence staining of the histological sections showed the presence of radial glia (SOX2) and mature neuronal (NeuN) cell types in iPSC-derived COs, which were also present in H9 hESC-derived COs ( Fig. 2D and Table S4 for replicates). These qualitative imaging results confirm successful in vitro differentiation of iPSCs derived from human PBMCs into COs. As a routine quality control to monitor the genomic integrity across the production stages (PBMCs, iPSC, iPSC-derived CO), we performed a simple sex characterization experiment and demonstrated no change to the female genomic DNA (Fig. S1-B, Tables S5 and S6), allowing us to use these COs for downstream investigation.
Mitochondrial genetics, function and morphology from PBMCs, iPSCs to COs. Mitochondrial function has been classified as a key indicator of neuronal activity and healthy cells. Thus, we next monitored the integrity and stability of mitochondrial genotype, function and morphology across the primary (PBMCs), reprogrammed (iPSCs), and differentiated (COs) stages (Table S4 for replicates).
Mitochondria harbours its own genome in multiple copies, called the mitochondrial DNA (mtDNA). The mtDNA encodes required subunits of the electron transport and oxidative phosphorylation complexes as well as the ribosomal and transfer RNAs required for their translation 18 . As a result, it is critical to evaluate whether mtDNA integrity is preserved throughout the process of generating COs 19 . The integrity of mtDNA can be evaluated by identifying the: (1) haplogroup, which is defined by a set of genetic variants associated with maternal ancestry; (2) percentage of heteroplasmy (mix of normal and mutated mtDNA) or homoplasmy (uniform collection of mtDNA, either mutated or normal) and; (3) copy number.
Using mtDNA sequencing, we identified the X2g haplogroup across PBMCs, iPSCs and iPSC-derived COs (Fig. 2E, Tables S7, S8). We further validated these results by performing polymerase chain reaction (PCR) amplification of short mtDNA fragments corresponding to the X2g haplogroup and sequencing these products (Table S9). No change to the mtDNA haplogroup was observed (Table S8), confirming that the mitochondrial ancestry of the donor was retained throughout the process of CO production. The mtDNA is also susceptible to oxidative damage and lack protective histones, which could potentially lead to novel mutations in the iPSCs and COs 20 . To ensure the preservation of mtDNA integrity throughout the CO production, we evaluated the complete mtDNA sequence and confirmed that the nucleotide identity across PBMCs, iPSCs and iPSC-derived COs is 100%. As no novel mutations were introduced, these results assure that we have retained the mtDNA identity of the donor during the reprogramming and differentiation process. A recurring question in the scientific community is whether there is a selection process that drives homoplasmy from heteroplasmy upon iPSC reprogramming [21][22][23] . Using the mtDNA sequencing data, we demonstrated the conservation of four heteroplasmic variants across PBMCs, iPSCs and iPSC-derived COs suggesting no selection process occurred (Fig. 2E). To further evaluate mtDNA integrity, we performed quantitative polymerase chain reaction (qPCR) using specific primers that target a highly conserved gene in the mtDNA (NADH dehydrogenase subunit 1, MT-ND1) and quantified the ratio of MT-ND1 gene copy to two copies of a single-copy nuclear gene (Beta-2-Microglobulin, b2M). Our results revealed multiple copies of mtDNA, which were preserved upon CO production from iPSCs (Fig. 2E, Table S10). Altogether, we provided solid evidence that the iPSC-derived COs retained the mtDNA genetic identity of their somatic origin (PBMC), resulting in the recapitulation of the donor's mitochondrial phenotype.
While mtDNA integrity was preserved, a remaining question is whether this translates to healthy and functional mitochondria. As a first approach, we stained COs before and after differentiation using MitoTracker Red CMXRos, which is a cationic fluorescent dye that penetrates solely into active and live mitochondria in a potential-dependent manner. Following sample staining, we demonstrated MitoTracker-positive cells across PBMCs, iPSCs and iPSC-derived COs, which were similarly detected in H9 hESCs and H9 hESC-derived COs (Fig. 3A). As the mitochondria appeared live and active, we next investigated function and responsiveness. The functional state of the mitochondria can be evaluated by monitoring changes in the mitochondrial membrane potential (MMP) 24 . JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimi-dazolylcarbocyanine iodide) is a dual emission cationic dye that accumulates in the mitochondria in response to the MMP, yielding a green fluorescence in less energetic mitochondria and red fluorescent aggregates in highly energetic mitochondria. Here, we used JC-1 dye as it is optimal for the end-point analysis of mitochondrial health 25 . Treatment of the H9 hESC-and iPSC-derived COs before and after differentiation with JC-1 led to an increased red-to-green fluorescence ratio, indicative of higher MMP, corresponding to a highly energetic mitochondrial state ( Fig. 3B and Fig. S2-A). We next investigated whether these COs, both before and after differentiation, remained responsive to pharmacological intervention. For this, we administered carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP), an uncoupler of oxidative phosphorylation (OXPHOS) that collapses and depolarizes the MMP to serve as our depolarization control. Treatment with FCCP led to a marked decrease in the red-to-green fluorescence ratio and fragmentation of the mitochondrial tubular network into smaller circular monomers ( Fig. 3C and Fig. S2-A), indicating that in all cases, the COs are responsive to pharmacological intervention. Overall, these data demonstrate that the mitochondria remained functional and responsive throughout the process of generating COs.
The MMP is generated upon the passing of electrons through a group of mitochondrial protein complexes I, III, and IV located on the inner mitochondrial membrane, which in turn is used to drive adenosine www.nature.com/scientificreports/ 5′-triphosphate (ATP) production through complex V. We determined the ability of COs derived from iPSCs and H9 hESCs to perform OXPHOS by measuring the assembly levels of intact mitochondrial complexes and the intracellular ATP levels. Using Luminex bead-based multiplex immunoassay, we successfully tracked the expression of intact mitochondrial complexes I-V across PBMC, iPSC and iPSC-derived COs and H9 hESC and H9 hESC-derived COs (Fig. 4A). To visualize the proper formation and distribution of OXPHOS, we performed immunofluorescence staining of TOMM-20, a marker of the outer mitochondrial membrane and NDUFS3, SDHA, UQCRC1, COX-IV, and ATP Synthase-β, markers of the inner mitochondrial membrane of complexes I, II, III, IV and V, respectively. We observed typical perinuclear mitochondrial distribution and expression (where clusters of mitochondria surround the nucleus) throughout the COs and cells ( Fig. 4B and Table S2). To evaluate endpoint ATP levels that result from the balance between ATP production and usage, we performed CellTiter-Glo luminescent assay and shown the maintenance of ATP levels throughout the generation of COs, which was in a similar pattern to that observed for the OXPHOS data indicating high levels of endpoint ATP in stem cells as compared to COs ( Fig. 4C and Fig. S2-B). Energy metabolism has been suggested to play a crucial role in the regulation and maintenance of pluripotency [26][27][28] . To examine this further, we used oligomycin, an inhibitor of complex V, to block ATP production by mitochondrial OXPHOS in PBMC-derived iPSCs and H9 hESCs. We observed a subtle decrease (~ 20%) in ATP levels ( Fig. S2-C), suggesting that the immediate source of ATP is glycolysis in iPSCs and H9 hESCs. Although less efficient in terms of energy production, stem cells have been shown to use this pathway at a much faster rate which is crucial for maintaining pluripotency and may explain the high ATP levels 26,27,29 . In contrast, 4-month-old H9 hESC COs treated with oligomycin had a ~ 55% reduction in intracellular ATP, suggesting that COs utilize both OXPHOS and glycolytic pathways as their source of energy to at a similar extent ( Fig. S2-C). Lastly, we monitored mitochondrial number and visualized morphology using transmission electron microscopy (TEM). Consistent with OXPHOS and ATP production, H9 hESCs and iPSCs had higher number of mitochondria compared to their corresponding COs ( Fig. 4D-i). Moreover, the electron micrographs revealed well-preserved mitochondrial morphologies with defined cristae before and after the generation of COs ( Fig. 4Dii). Consistent with expectations, mitochondria in the iPSCs and H9 hESCs displayed globular and immature shapes with electron-dense cristae whereas more elongated and mature mitochondria with thinner cristae were visually observed in the COs (Fig. 4D-ii), suggesting mitochondrial maturation during the differentiation process. While mature mitochondrial morphology was observed in COs, the source of ATP generation was not entirely dependent on OXPHOS ( Fig. S2-C), suggesting that metabolic shift may follow as the COs becomes progressively more mature.
Electrophysiological responses in iPSC-derived cerebral organoids. A major functional test of any neuronal preparation is the ability to form functional mature neurons with active synaptic neurotransmission 30 . These properties can be tested using electrophysiological recordings from individual neurons and analysis of their action potential (AP)-generation and synaptic activity. Whole-cell recordings were performed in acute slices prepared from COs derived from iPSCs and H9 hESCs ( Fig. 5 and Table S4 for replicates). All recorded neurons (N = 26 cells for H9 hESC-derived COs and N = 29 cells for iPSC-derived COs) were divided into three types based on a combination of electrophysiological properties, specifically immature neurons (Type 1), developing neurons (Type 2), and mature neurons (Type 3). Type 1 neurons were immature, could not generate APs, had higher membrane resistance and lower capacitance (Table S11), and relatively small sodium and potassium currents triggered by depolarization (Fig. 5B). Spontaneous activity was not recorded in type 1 immature neurons. Both type 2 developing neurons and type 3 mature neurons were able to generate APs; however, type 2 developing neurons had smaller amplitude and slower kinetics when compared to type 3 mature neurons (Fig. 5A). Correspondingly, sodium and potassium currents were smaller in type 2 developing neurons compared to type 3 mature neurons (Fig. 5B). In addition, only type 3 mature neurons generated stable trains of  Table S8. www.nature.com/scientificreports/ spontaneous APs at holding membrane level or during slight depolarization (Fig. 5C). However, spontaneous AP frequency and synaptic activity were similar between type 2 developing neurons and type 3 mature neurons (Fig. 5C). Next, the electrophysiological properties of type 2 developing neurons and type 3 mature neurons in COs generated from PBMC-derived iPSCs were compared to those found in H9 hESC-derived COs. We found similar AP amplitude (  (B) (i) Bar graph summarizing mitochondrial membrane potential (MMP) as red-to-green fluorescence ratio across primary, reprogrammed and differentiated stages in samples treated with JC-1 only (control basal MMP level) and those treated with carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP). Fluorescence was recorded using a microplate reader. Bars, mean ± SD; (ii) Representative MMP images of a fully captured neuron with cell body and axon projection in H9 hESC CO (left, scale bar, 5 μm) and iPSC CO (right, scale bar, 7 μm). Red, JC-1 aggregates (highly energetic or polarized), green, JC-1 monomers (less energetic or depolarized). (C) (i) Top row shows representative MMP images across CO generation from H9 hESCs (scale bar, 25 μm) to H9 hESC CO (scale bar, 25 μm) or hESC CO single cell (SC, scale bar, 10 μm) and; (ii) from PBMCs (scale bar, 25 μm) to iPSCs (scale bar, 25 μm) to iPSC COs (scale bar, 25 μm) or iPSC CO SC (scale bar, 5 μm). Bottom row shows representative images of samples treated with FCCP followed by JC-1 to visualize the collapse of MMP in (i) H9 hESCs (scale bar, 25 μm) to H9 hESC CO (scale bar, 25 μm) or hESC CO SC (scale bar, 5 μm) and; (ii) from PBMCs (scale bar, 25 μm) to iPSCs (scale bar, 25 μm) to iPSC COs (scale bar, 25 μm) or iPSC CO SC (scale bar, 5 μm). All images were captured at ×100 magnification with slight variations in the zoom factor to capture the single cell(s) or single colony of interest. Immunofluorescent images shown here were used for qualitative observations only.

Discussion
To the best of our knowledge, this is the first report demonstrating the development of a functional humanderived CO model made from PBMCs successfully tracking overall mitochondrial health, including genetics, function and morphology. Although the COs are immature, more closely resembling a fetal rather than an adult brain [31][32][33][34] , they exhibited electrophysiological responses including both mature and immature neurons and displayed functional and responsive mitochondria across all stages of development, from PBMCs to iPSCs to iPSCderived COs. We demonstrated the preservation of the donor's mtDNA genetic integrity, highlighting the potential of COs generated from PBMC-derived iPSCs to recapitulate the functional phenotypes that may be rooted in their genetic information. We further demonstrated the response of iPSC-derived COs to pharmacological ; complex II, SDHA; complex III, UQCRC1; complex IV, COXIV; complex V, ATP synthase-β; green) and the outer mitochondrial membrane (TOMM-20; red) in (i) H9 hESCs to H9 hESC COs and; (ii) iPSCs to iPSC COs. All scale bars, 100 μm. Last column shows insets, enlarged views of boxed areas from the merge CO images, all scale bars, 10 μm. In the CO tissues, out of focus light and autofluorescence of tissue matrix led to background noise which were corrected for better visualization. Brightness levels of each images were also adjusted to optimize visualization. Immunofluorescent images shown here were used for qualitative observations only-no quantitative analyses were performed. www.nature.com/scientificreports/ treatment (FCCP), mimicking the individual's overall cellular/mitochondrial response, which supports the use of iPSC-derived COs as a representative model to evaluate function and to screen for therapeutic compounds. Despite encouraging progress, CO models do have limitations. An ongoing challenge is that with prolonged culture, COs reach a size limit along with the occurrence of cell death in the centre regions due to insufficient diffusion of oxygen and nutrients 11,35 . While it may be possible to increase oxygen availability by growing COs in a higher oxygen environment, this can be toxic if not carefully controlled 36 . So far, COs do not develop vasculature that could provide nutrients, but studies involving vascularization, generation of blood vessel organoids, and the development of microfluidics are underway and have yielded some promising results [37][38][39] . With these technological advancements, we expect that mitochondrial maturation will follow during the CO maturation process and become progressively more OXPHOS dependent on energy production. Without these technological advancements, COs remain immature and do not mature past the prenatal stage. As such, the COs currently on the research market are only beneficial in investigating mitochondrial dysfunction during early neurodevelopment in the brain of diseases that may have an underlying neurodevelopmental origin.
Furthermore, the long-term culture required for the maturation of COs to 4.5 months limits the amount of material available for screening. This issue can delay the clinical process of diagnostic and therapeutic development and turnaround time. Thus, future studies are needed to develop methods that will allow these organoids to fully and rapidly maturate into an adult brain to allow for high throughput mitochondrial diagnostics and therapeutic development in a clinical context. Next, we only evaluated mitochondrial health at the whole tissue level at one time point as an initial proof-of-concept; however, energy production and consumption can vary amongst different cell types in the brain. As a result, future studies are needed to sort the cells and evaluate mitochondrial function in different cell types within the COs and at other time points. Finally, we are aware that respirometry approach is the gold standard and widely used to evaluate mitochondrial function in cells. However, respirometry techniques are currently standardized in two-dimensional single-cell systems and not three-dimensional COs above 500 μm in size due to the size limitations of the measurement chamber (Agilent Technologies, Inc). While using single cells from dissociated COs has been proposed, the loss of three-dimensional interconnection between neurons and different cell types, as well as subsequent single cell culturing can affect the viability and cellular behaviour, which in turn can affect the interpretation of the data. With these limitations in mind, future work is warranted to overcome these technological limitations.
While acknowledging that CO models currently do not have the full precision of a human brain, the ability to interrogate overall mitochondrial health in a human context is extremely valuable for studying brain development and diseases. By evaluating mitochondrial health in both the PBMCs and iPSC-derived COs, we can start to identify changes in the brain that may also be present in the blood of patients. As a result, this approach can aid in the development of biomarkers, companion diagnostics, and novel mitochondrial therapeutics that can inform critical clinical decisions in mitochondrial medicine. As there is currently no standard of care for the assessment of mitochondrial health in both the brain and peripheral blood of patients, the platform provided here is a potential starting point that can be applied to a wide range of human diseases.

Materials and methods
Blood sample collection and processing. We followed the guidelines established by the Biomarkers Task Force as modified by the World Federation of Societies of Biological Psychiatry for clinical assessment and documentation, ethical procedures and blood sample collection 40 . This study was performed in accordance with the latest version of the Declaration of Helsinki and approved by the Research Ethics Board at the University of Toronto, Canada (Protocol Number: 29949). Informed consent of the participant was obtained after the nature of the procedures had been fully explained. Venous blood (10 mL) was drawn from a participant through venipuncture into a plastic whole-blood tube with spray-coated K 2 EDTA (16 × 100 mm × 10.0 mL BD Vacutainer Plus). Whole blood was carefully layered on Ficoll-Paque (GE Healthcare, 17144002) at a 1:1 ratio in a conical tube and centrifuged at 400×g for 40 min. Following centrifugation, peripheral blood mononuclear cells www.nature.com/scientificreports/ that exhibited embryonic stem cell (ESC)-like morphologies were picked under a stereomicroscope for expansion and maintenance in Essential 8 medium (Gibco, A1517001). Eight drops of Carnoy's fixative (methanol/ acetic acid, 3:1) were added and mixed together. Cells were centrifuged at 200×g for 10 min, supernatant was aspirated until 0.5 mL remained, and cell pellet was flicked to resuspend completely. Carnoy's fixative was added to the 14 mL mark, tubes were inverted to mix, and incubated in − 20 °C for at least one hour to overnight. After two more rounds of fixations (add 8 mL fixative, invert to mix, and centrifuge at 200×g for 10 min), cells were resuspended in 0.5-1 mL of fixative and cells from each suspension were dispensed onto glass slides and baked at 90 °C for 1.5 h, followed by overnight aging at room temperature in desiccator. Routine G-banding analysis was then carried out. Twenty metaphases per cell line were examined.

Characterization of induced pluripotent stem cells. We followed the workflow developed by
Generation of cerebral organoids from H9 hESCs and iPSCs. Cerebral organoids were generated from iPSC and H9 hESCs as previously described 17 . In brief, stem cells were aggregated into embryoid bodies and after induction of the neural germ layer were embedded in Matrigel droplets in differentiation media. Once removed from the Matrigel, free-floating organoids were maintained in prolonged culture on an orbital shaker.

Fluorescent immunohistochemistry for the characterization of cerebral organoids. Whole
COs derived from H9 hESCs and iPSCs were fixed with 4% paraformaldehyde at 4 °C overnight on an orbital shaker, cryoprotected in 30% (weight/volume) sucrose at 4 °C overnight and embedded in optimal cutting temperature compound. Data were filtered at 4 kHz, and acquired using pClamp 10 software (Molecular devices, Sunnyvale, CA, USA). All recordings were done at a holding potential − 70 mV. The uncompensated series resistance was monitored by the delivery of − 10 mV steps throughout the experiment, only recordings with less than 15% change were analyzed. Resting membrane potential was measured immediately after establishing whole-cell patch clamp recording and followed by measurements of passive neuronal properties (access resistance, membrane resistance and capacitance) using automatic membrane test in pClamp 10 software. Ability to generate spontaneous action potentials was tested in current clamp mode starting from holding membrane potential and by application of depolarizing steps (5 pA increments) until steady firing was reached. This steady firing was observed only in mature neurons. Voltage-dependent sodium and potassium currents were measured in voltage clamp mode, using 500 ms voltage steps applied from a holding potential to a range of potentials between − 50 and + 50 mV (in 10 mV increments). Action potentials were triggered using 500 ms depolarizing pulses increasing amplitude (in 5 pA steps). Synaptic events were analyzed using pClamp 10 software within 5 min of recordings, individual events were detected using automatic template search. Templates were created using the average of at least 10 events aligned by the rising of their slopes. AP amplitude, rise time, half width and time to peak (TTP) were calculated to investigate excitability. See Table S4 for biological and technical replicates.

Total DNA extraction. Genomic DNA was isolated from PBMCs, stem cells (H9 hESCs and iPSCs) and
COs derived from H9 hESCs and iPSCs using the QIAamp DNA Mini Kit (Qiagen, 51304) by following the manufacturer's recommended protocol and using the recommended elution volumes. The concentration, purity and integrity of all extracted DNA was determined by spectrophotometric measurement on the NanoDrop ND-1000 (Thermo Fisher Scientific). www.nature.com/scientificreports/ Mitochondrial DNA sequencing for haplogroup and heteroplasmy characterization. The mitochondrial DNA was amplified as two fragments using two the following two primer sets: (1) COIII-F (5′-TCA  CAA TTC TAA TTC TAC TGA-3′) and mt16425R (5′-GAT ATT GAT TTC ACG GAG GAT GGT G-3′) resulting in a 7321 bp fragment and (2) mt16426F (5′-CCG CAC AAG AGT GCT ACT CTC CTC -3′) and COIII-R (5′-CGG ATG AAG CAG ATA GTG AGG-3′) and 10,011 bp products. PCR reactions were performed using TaKaRa LA Taq Hot Start polymerase kit (TaKaRa) and 50 ng of total genomic DNA in a 50 µL PCR reaction. Cycling conditions included an initial denaturation at 94 °C for 2 min, followed by 30 cycles of denaturation at 94 °C for 20 s, annealing at 60 °C for 30 s, and extension at 68 °C for 12 min. The reaction was concluded with a final extension at 68 °C for 20 min. PCR products were analyzed on an agarose gel and HindIII Ladder (New England Biolabs), and subsequently purified with the GeneJet PCR Purification kit (ThermoFisher). Samples were then submitted for Illumina sequencing Nextera XT library prep on a Hiseq 2500 high throughput flowcell 2 × 125 bp.
Mitochondrial DNA copy number. We adapted a protocol from Picard et al. for measuring relative mtDNA copy number by qPCR 42 . Relative mtDNA copy number was measured by calculating the ratio of MT-ND1 gene copy to two copies of a single-copy nuclear gene (β-2 microglobulin). As mtDNA quantification is relative (not absolute), the results presented here do not represent actual copy numbers. Briefly, total genomic DNA was extracted as described in the method "Total DNA Extraction" and diluted to 0.1 ng/μL. The ratio of mtDNA to nuclear DNA was quantified by 2 −ΔCt method using the following primer pairs: β-2 microglobulin forward (TGC TGT CTC   ). An ATP standard curve was generated using ATP disodium salt (Sigma-Aldrich, A7699) ranging from 0 to 1 μM with 100 μL of 1 μM ATP solution containing 10 -10 mol ATP. After addition of CellTiter-Glo Reagent (100 μL) to each well, contents were mixed on an orbital shaker for 2 min and luminescence readings from experimental samples and ATP standards were acquired using a Synergy H1 microplate reader (BioTek Instruments, Inc., 253147) and Gen5 Software. See Table S4 for biological and technical replicates.
Oligomycin treatment. To confirm whether the main source of ATP production is from OXPHOS or glycolysis in iPSCs and H9 hESCs, cells were first seeded at a density of 155,000 cells/cm 2 or 1.5 million cells/ well in a clear 6-well plate format. Cells were visualized 24 h later to ensure proper attachment and health of the cells. Then, we treated the cells with 1 μM of Oligomycin for 30 min in MEF-conditioned media with a base DMEM/F12 that contains energy substrates, including high glucose and sodium pyruvate (Life Technologies, #11330057) 17 . The concentration and time of Oligomycin have been established in many Agilent Seahorse assays and and human pluripotent stem cell energy-profiling studies to inhibit OXPHOS-linked ATP production by blocking complex V, without inducing cell death in stem cells and cultured neurons 27,28,48 . Following treatment, cells were detached using Gentle Cell Dissociation Reagent (STEMCELL Technologies Inc) and propidium iodide staining on the Orflo Moxi Flow were used to assess cell count and viability. Cells were then seeded at a density of 50,000 cells per 100 μL culture media per well in a 96-well white polystyrene plate (155,000 cells/cm 2 , Greiner CELLSTAR, 655083) and lysed immediately with CellTiter-Glo Reagent for endpoint ATP measurement as described above. As proof-of-concept, we treated whole H9 hESC COs (4 month) using Oligomycin and seeded the cells for endpoint ATP measurement as described above.
Luminex technology multiplex human oxidative phosphorylation (OXPHOS) magnetic bead panel. The assembly of mitochondrial complexes I to V in PBMCs, stem cells (H9 hESCs and iPSCs) and COs derived from H9 hESCs and iPSCs were measured using the Human Oxidative Phosphorylation Magnetic Bead Panel Milliplex MAP Kit (Millipore-H0XPSMAG-16K), following the manufacturer's instructions. Briefly, cell or tissue pellets were resuspended in Cell/Mitochondrial Lysis Buffer (as recommended by Millipore) with protease inhibitors (1:100, EMD Chemicals, Catalog #535140) and phosphatase inhibitors (1:50, EMD Chemicals, Catalog #524629). Protein lysates (500 μg/mL) were incubated with specific proprietary capture antibodies raised against the assembled OXPHOS protein complexes (I, II, III, IV and V) for 2 h at room temperature. The samples were then incubated with biotinylated secondary detection antibodies for 1 h at room temperature followed by incubation with streptavidin phycoerythrin conjugate for 30 min, which is the reporter molecule to complete the reaction. Samples were ran in five technical replicates. The intensity of the fluorescent signal was acquired using the Luminex Magpix system (Luminex Corporation xMAP Technology) and xPONENT software and analyzed using the MILLIPLEX Analyst 5.1 software. Results were expressed as normalized median fluorescence intensity. See Table S4  www.nature.com/scientificreports/ 1:500) and TOMM20 (rabbit, abcam, ab186734, 1:100) with specific combinations (NDUFS3 + TOMM20, SDHA + TOMM20, UQCRC1 + TOMM20, COXIV + TOMM20 and ATP Synthase β + TOMM20) diluted in antibody blocking solution (1X PBS pH 7.4, 0.1% Tween-20, 1% BSA, and 5% Normal Donkey Serum) and incubated overnight at 4 °C. Following overnight incubation, samples were washed three times (5 min per wash) with 0.1% Tween-20 in 1X PBS pH 7.4 and then incubated overnight at 4 °C (for tissues) or 1 h at room temperature (for cells) with the appropriate secondary antibodies: Donkey anti-Mouse IgG (H + L) Highly Cross-Adsorbed Secondary Antibody Alexa Fluor Plus 488 (Invitrogen, A23766) and Donkey anti-Rabbit IgG (H + L) Highly Cross-Adsorbed Secondary Antibody Alexa Fluor Plus 647 (Invitrogen, A32795) diluted 1:750 in antibody blocking solution. Following secondary antibody incubation, samples were washed three times in 1X PBS pH 7.4 with 0.1% Tween-20. Samples were counterstained with ProLong Gold Antifade Mountant with DAPI (Invitrogen) and left to cure overnight at room temperature protected from light. Images were captured using Leica TCS SP8 lightning confocal laser scanning microscope (DMI6000) equipped with white light laser (470-670 nm) and collected using LASX software (Lecia Microsystems). See Table S4 for biological and technical replicates.
Transmission electron microscopy. Mitochondrial number and morphology for PBMCs, stem cells (H9 hESCs and iPSCs) and COs derived from H9 hESCs and iPSCs were evaluated using Transmission Electron Microscopy. Samples (PBMCs, H9 hESCs, iPSCs, and COs) were fixed with primary fixation buffer (0.1 M phosphate buffer pH 7.2, 4% paraformaldehyde, 1% glutaraldehyde) for 2 h at room temperature and replaced with fresh fixative overnight at 4 °C. Following overnight incubation, samples (PBMCs, H9 hESCs, iPSCs, and COs) were washed three times for 20 min each with 0.1 M phosphate buffer at room temperature and underwent a secondary fixation with 1% osmium tetroxide in 0.1 M phosphate buffer for 1 h at room temperature in the dark. The samples (PBMCs, H9 hESCs, iPSCs, and COs) were again washed with 0.1 M phosphate buffer (pH 7.2) three times for 10 min each. Dehydration steps were performed on the samples by using a graded series of ethanol (EtOH) and distilled water at room temperature: 50% EtOH twice for 10 min each, 70% EtOH twice for 10 min each, 90% EtOH twice for 10 min each, and 100% EtOH three times for 15 min each. Once samples (PBMCs, H9 hESCs, iPSCs, and COs) were dehydrated, they were washed with a transitional solvent, propylene oxide, twice for 15 min each. The samples (PBMCs, H9 hESCs, iPSCs, and COs) were then infiltrated with epon resin using a graded series of epon and propylene oxide mixture at room temperature: (1) one part Epon resin mixed with two parts 100% propylene oxide for 1 h using an agitator; (2) two parts Epon resin mixed with one part 100% propylene oxide for 3 h using an agitator; (3) 100% epon overnight using an agitator and finally; (4) fresh 100% epon resin for 2 h. Once infiltration was complete, the samples (PBMCs, H9 hESCs, iPSCs, and COs) were placed in a Beem embedding capsule and polymerized at 60 °C for 48 h. After complete polymerization, the solid resin block containing the samples were sectioned on a Reichert Ultracut E microtome to 90 nm thickness and collected on 200 mesh copper grids. The sections were stained using saturated uranyl acetate for 15 min, rinsed in distilled water, followed by Reynold's lead citrate for 15 min and rinsed again in distilled water. The sections were examined and photographed using a FEI Talos L120C transmission electron microscope at an accelerating voltage of 80 kV.