Stable transplantation of human mitochondrial DNA by high-throughput, pressurized isolated mitochondrial delivery

  1. Alexander J Sercel
  2. Alexander N Patananan
  3. Tianxing Man
  4. Ting-Hsiang Wu
  5. Amy K Yu
  6. Garret W Guyot
  7. Shahrooz Rabizadeh
  8. Kayvan R Niazi
  9. Pei-Yu Chiou
  10. Michael A Teitell  Is a corresponding author
  1. Molecular Biology Interdepartmental Doctoral Program, University of California, Los Angeles, United States
  2. Department of Pathology and Laboratory Medicine, David Geffen School of Medicine, University of California, Los Angeles, United States
  3. Department of Mechanical and Aerospace Engineering, University of California, Los Angeles, United States
  4. NanoCav, LLC, United States
  5. NantBio, Inc, and ImmunityBio, Inc, United States
  6. NantOmics, LLC, United States
  7. California NanoSystems Institute, University of California, Los Angeles, United States
  8. Department of Bioengineering, University of California, Los Angeles, United States
  9. Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research University of California, Los Angeles, United States
  10. Department of Pediatrics, David Geffen School of Medicine, University of California, Los Angeles, United States
  11. Jonsson Comprehensive Cancer Center, David Geffen School of Medicine, University of California, Los Angeles, United States
4 figures, 1 table and 1 additional file

Figures

Figure 1 with 1 supplement
Pressure simulations of mitochondrial transfer tools.

(A) Schematic of MitoPunch apparatus. Recipient cells (1 × 105) are seeded on a porous polyester (PET) membrane ~24 hr before delivery. A freshly isolated suspension of mitochondria in 1× Dulbecco's Phosphate Buffered Saline (DPBS) with calcium and magnesium, pH 7.4, is loaded into the polydimethylsiloxane (PDMS) chamber and the filter insert is sealed over the PDMS before activation of the mechanical plunger to pressurize the apparatus and deliver the mitochondrial suspension into recipient cells. (B) Numerical simulation showing the pressure inside the PDMS chamber reaching 28 kPa with piston activation. COMSOL file used to model MitoPunch pressure is available in Figure 1—source data 1. (C) Schematic of MitoCeption technique. Recipient cells (1 × 105) are seeded on wells of a 6-well dish ~24 hr before delivery. A freshly isolated suspension of mitochondria in 1× DPBS with calcium and magnesium, pH 7.4, is pipetted into the cell medium before the plate is centrifuged at 1500 × g for 15 min at 4°C. The plate is incubated in a 37°C incubator for 2 hr before being centrifuged again at 1500 × g for 15 min at 4°C. (D) MitoCeption pressure model and calculated pressure exerted by isolated mitochondria on recipient cells during delivery.

Figure 1—source data 1

Numerical simulation of MitoPunch pressure generation during mitochondrial delivery.

https://cdn.elifesciences.org/articles/63102/elife-63102-fig1-data1-v2.zip
Figure 1—figure supplement 1
Annotated MitoPunch apparatus.

Annotated image of the MitoPunch apparatus. Labeled parts are described in the Materials and methods to assist with construction of the apparatus.

Figure 2 with 1 supplement
MitoPunch delivers isolated mitochondria to recipient cells.

(A) Quantification of flow cytometry results measuring the association of dsRed mitochondria with 143BTK– ρ0 and BJ ρ0 single recipient cells following mitochondrial transfer. (B) Mean and median dsRed spot count quantification of ImageStream data. (C) Sequential Z-stacks of confocal microscopy of 143BTK– ρ0 cells delivered isolated HEK293T-derived dsRed mitochondria by coincubation, MitoPunch, and MitoCeption and fixed 15 min following transfer. Arrows indicate representative mitochondria interacting with recipient cells. Transferred dsRed mitochondria are labeled in red. Plasma membranes are labeled in green, stained with CellMask Green plasma membrane stain in coincubation and MitoCeption and with wheat germ agglutinin plasma membrane stain in MitoPunch. Scale bars indicate 15 µm. (D) Quantification of flow cytometry measurements of fluorescence in 143BTK– ρ0 and BJ ρ0 single cells following propidium iodide transfer by coincubation, MitoPunch, and MitoCeption. Error bars represent SD of three technical replicates in all figures.

Figure 2—figure supplement 1
Mitochondrial spot quantification.

Representative spot count distributions, bright-field images, and PE channel fluorescent images from ImageStream imaging flow cytometry representing the number of dsRed spots associated with 143BTK– ρ0 and BJ ρ0 cells 2 hr after mitochondrial transfer by coincubation, MitoPunch, and MitoCeption. Imaging flow cytometry data is represented as histograms normalized to the mode of each data set. Scale bars indicate 10 µm.

Figure 3 with 6 supplements
Stable retention of transplanted mitochondrial DNA (mtDNA) into transformed and replication-limited cells.

(A) Workflow for stable isolated mitochondrial recipient (SIMR) cell generation by mitochondrial transfer into ρ0 cells. (B) Representative fixed and crystal violet stained 10 cm plate image following MitoPunch and SIMR cell selection used for SIMR clone generation quantification. (C) Quantification of crystal violet stained 143BTK– ρ0 and BJ ρ0 SIMR clones. Error bars represent SD of three technical replicates. (D) Quantification of crystal violet stained 143BTK– ρ0 and BJ ρ0 SIMR clones formed by MitoPunch actuated with indicated voltages after uridine-free selection. Error bars represent SD of three technical replicates with the exception of BJ ρ0 5 V transfer, which shows two replicates. (E) Quantification of crystal violet stained 143BTK– ρ0 and BJ ρ0 SIMR clones formed by MitoCeption with indicated centripetal forces after uridine-free selection. Error bars represent SD of three technical replicates.

Figure 3—figure supplement 1
Verification of surviving mitochondrial donor cells following mitochondrial isolation.

Images of three crystal violet stained 10 cm plates seeded with isolated mitochondria from ~1.5 × 10⁷ HEK293T dsRed donor cells taken from three independent mitochondrial isolations following dialyzed medium selection. Pictures were taken on a circular white disk matted within a cardboard frame for clarity.

Figure 3—figure supplement 2
MitoPunch generates stable isolated mitochondrial recipient (SIMR) clones in immortalized mouse cells.

Quantification of crystal violet stained B16 ρ0 SIMR clones formed by MitoPunch transfer of isolated L929 mitochondria actuated with indicated voltages after SIMR cell selection. Error bars indicate the range between the technical duplicates.

Figure 3—figure supplement 3
Quantification of MitoPunch reproducibility.

Quantification of crystal violet stained 143BTK– ρ0 stable isolated mitochondrial recipient (SIMR) clones generated in technical triplicate from three independent HEK293T dsRed mitochondrial donor cell preparations plotted alongside technical singlet DPBS delivery and plated mitochondrial preparation controls (Figure 3—figure supplement 1).

Figure 3—figure supplement 4
Quantification of MitoPunch reproducibility relative to mitochondrial mass transferred.

Quantification of crystal violet stained 143BTK– ρ0 stable isolated mitochondrial recipient (SIMR) clones generated in technical triplicate from three independent HEK293T dsRed mitochondrial donor cell preparations plotted as number of SIMR clones generated per µg isolated mitochondrial protein loaded into the polydimethylsiloxane (PDMS) reservoir.

The mass of isolated mitochondria per 120 µL of isolated mitochondrial suspension for the three preparations are as follows: Prep 1–27 µg, Prep 2–33 µg, Prep 3–25 µg.

Figure 3—figure supplement 5
Quantification of stable isolated mitochondrial recipient (SIMR) generation efficiency by delivering different masses of isolated mitochondria.

Quantification of crystal violet stained 143BTK– ρ0 SIMR clones using indicated concentrations of mitochondrial suspension following 7 days of culture in SIMR selection medium. Error bars represent SD of three technical replicates.

Figure 3—figure supplement 6
Quantification of MitoPunch stable isolated mitochondrial recipient (SIMR) generation by serial deliveries using one isolated mitochondrial aliquot.

Quantification of crystal violet stained SIMR clones formed by serial MitoPunch deliveries of HEK293T dsRed mitochondria into 143BTK– ρ0 recipient cells using the same used mitochondrial sample remaining in the polydimethylsiloxane (PDMS) reservoir after the preceding delivery.

Figure 4 with 2 supplements
Mitochondrial DNA (mtDNA) transplantation rescues ρ0 mitochondrial phenotypes.

(A) Oxygen consumption rate (OCR) quantification of basal and maximal respiration, spare respiratory capacity, and ATP generation from two independent 143BTK– ρ0 + HEK293T stable isolated mitochondrial recipient (SIMR) clones generated by MitoPunch and MitoCeption. Cross-hatched data indicate clones that were frozen and thawed twice each. Error bars represent SD of four technical replicates for fresh SIMR cell measurements and five for thawed SIMR cell measurements. (B) Confocal microscopy of representative 143BTK– ρ0 + HEK293T SIMR clones compared to 143BTK– parental, HEK293T dsRed mitochondrial donor, and 143BTK– ρ0 controls. Mitochondria were stained with anti-TOM20 antibody and labeled red, double-stranded DNA was stained with anti-dsDNA antibody and labeled green, and cell nuclei were stained with NucBlue (Hoechst 33342) and labeled blue. Scale bars indicate 15 µm.

Figure 4—figure supplement 1
Schematic of the Seahorse Mito Stress Test.

Annotated plot of oxygen consumption as a function of time, including the identity and timing of drugs injected over the course of a Seahorse Mito Stress Test measurement. Annotations indicate how OXPHOS parameters presented in Figure 4A are quantified.

Figure 4—figure supplement 2
Confocal microscopy of stable isolated mitochondrial recipient (SIMR) lines.

Confocal microscopy of SIMR clones formed in 143BTK– ρ0 cells with 143BTK– parental, HEK293T dsRed mitochondrial donor, and 143BTK– ρ0 controls. The MitoCeption SIMR clone data on the left represents the SIMR line that lost respiration following freeze-thaw. Mitochondria were stained with anti-TOM20 antibody and labeled red, double-stranded DNA was stained with anti-dsDNA antibody and labeled green, and cell nuclei were stained with NucBlue (Hoechst 33342)and labeled blue. The 143BTK–, 143BTK– ρ0, and HEK293T dsRed control images are the same images used in Figure 4B. Scale bars indicate 15 µm.

Tables

Key resources table
Reagent type
(species) or resource
DesignationSource or referenceIdentifiersAdditional information
Cell line (Homo sapiens)143 BTK– ρ0 osteosarcomaPatananan et al., 2020,
ATCC
Cat. #CRL-8303; RRID:CVCL_9W36
Cell line (Homo sapiens)143 BTK– osteosarcomaATCCCat. #CRL-8303; RRID:CVCL_9W36
Cell line (Homo sapiens)BJ ρ0 foreskin fibroblast (male)Patananan et al., 2020
ATCC
Cat. #CRL-2522; RRID:CVCL_3653
Cell line (Homo sapiens)HEK293T dsRedMiyata et al., 2014A gift from the laboratory of Dr. Carla Koehler
Cell line (M. musculus)B16 ρ0 melanomaDong et al., 2017A gift from the laboratory of Dr. Michael Berridge
Cell line (M. musculus)L929 fibroblastsATCCCat. #CCLl-1
AntibodyAnti-TOMM20 (Rabbit monoclonal)AbcamCat. #ab78547
RRID:AB_2043078
IF(1:1000)
AntibodyAnti-dsDNA (Mouse monoclonal)AbcamCat. #ab27156
RRID:AB_470907
IF(1:1000)
AntibodyAnti-rabbit IgG (Donkey polyclonal)Thermo Fisher ScientificCat. #A-31573
RRID:AB_2536183
IF(1:100)
AntibodyAnti-mouse IgG (Donkey polyclonal)Thermo Fisher ScientificCat. #A-21202
RRID:AB_141607
IF(1:100)
Commercial assay or kitQproteome Mitochondria Isolation kitQiagenCat. #37612
Commercial assay or kitBCA protein assayThermo FisherCat. #23225
Chemical compound, drugPropidium iodideThermo Fisher ScientificCat. #P1304MP
Chemical compound, drugAccutaseThermo Fisher ScientificCat. #A1110501
Chemical compound, drug16% paraformaldehydeThermo Fisher ScientificCat. #28906
Chemical compound, drugTriton-X 100SigmaCat. #X100
Chemical compound, drugProLong Gold Antifade Mountant with DAPIInvitrogenCat. #P3691
Chemical compound, drugProLong Glass Antifade Mountant with NucBlue StainThermo Fisher ScientificCat. #P36985
Chemical compound, drugUridineThermo Fisher ScientificCat. #AC140770250
Chemical compound, drugGalactoseSigma-AldrichCat. #G5388-100G
Chemical compound, drugCellMask Green PMMolecular ProbesCat. #C37608
Chemical compound, drugAlexa Fluor 488 conjugated Wheat Germ AgglutininInvitrogenCat. #W11261
Chemical compound, drugCrystal violetThermo Fisher ScientificCat. #C581-25
Software, algorithmWave 2.6.2AgilentRRID:SCR_014526
Software, algorithmFlowJo 10.6.2BD BiosciencesRRID:SCR_008520
Software, algorithmIDEAS 6.2Luminex
Software, algorithmMultiphysics 5.3COMSOLRRID:SCR_014767
Software, algorithmImaris Viewer 9.5.1Oxford InstrumentsRRID:SCR_007370
Software, algorithmImaris File Converter 9.5.1Oxford InstrumentsRRID:SCR_007370
Software, algorithmPrism v.8GraphpadRRID:SCR_002798
Software, algorithmLAS X Lite 3.7.1.21655Leica
Software, algorithmFIJISchindelin et al., 2012
OtherDialyzed FBSLife TechnologiesCat#26400–044
Other12–well 3.0 µm Transparent PET MembraneCorningCat#353181
OtherGlass coverslipsZeissCat#474030–9000
OtherV3 96-well plateAgilentCat#101085–004
OtherVariable voltage MitoPunch apparatusImmunityBioInquiries regarding this device can be made to the corresponding author

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  1. Alexander J Sercel
  2. Alexander N Patananan
  3. Tianxing Man
  4. Ting-Hsiang Wu
  5. Amy K Yu
  6. Garret W Guyot
  7. Shahrooz Rabizadeh
  8. Kayvan R Niazi
  9. Pei-Yu Chiou
  10. Michael A Teitell
(2021)
Stable transplantation of human mitochondrial DNA by high-throughput, pressurized isolated mitochondrial delivery
eLife 10:e63102.
https://doi.org/10.7554/eLife.63102