Author response: Stable transplantation of human mitochondrial DNA by high-throughput, pressurized isolated mitochondrial delivery (2020)

Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Generating mammalian cells with specific mitochondrial DNA (mtDNA)–nuclear DNA (nDNA) combinations is desirable but difficult to achieve and would be enabling for studies of mitochondrial-nuclear communication and coordination in controlling cell fates and functions. We developed 'MitoPunch', a pressure-driven mitochondrial transfer device, to deliver isolated mitochondria into numerous target mammalian cells simultaneously. MitoPunch and MitoCeption, a previously described force-based mitochondrial transfer approach, both yield stable isolated mitochondrial recipient (SIMR) cells that permanently retain exogenous mtDNA, whereas coincubation of mitochondria with cells does not yield SIMR cells. Although a typical MitoPunch or MitoCeption delivery results in dozens of immortalized SIMR clones with restored oxidative phosphorylation, only MitoPunch can produce replication-limited, non-immortal human SIMR clones. The MitoPunch device is versatile, inexpensive to assemble, and easy to use for engineering mtDNA–nDNA combinations to enable fundamental studies and potential translational applications. eLife digest Mitochondria are specialized structures within cells that generate vital energy and biological building blocks. Mitochondria have a double membrane and contain many copies of their own circular DNA (mitochondrial DNA), which include the blueprints to create just thirteen essential mitochondrial proteins. Like all genetic material, mitochondrial DNA can become damaged or mutated, and these changes can be passed on to offspring. Some of these alterations are linked to severe and debilitating diseases. Both the double membrane of the mitochondria and their high number of DNA copies make treating such diseases difficult. A successful therapy must be capable of correcting almost every copy of mitochondrial DNA. However, the multiple copies of mitochondrial DNA create a problem for genetic research as current techniques are unable to reliably introduce particular mitochondrial mutations to all types of human cells to investigate how they may alter cell function. Sercel, Patananan et al. have developed a method to deliver new mitochondria into thousands of cells at the same time. This technique, called MitoPunch, uses a pressure-driven device to propel mitochondria taken from donor cells into recipient cells without mitochondrial DNA to reestablish their function. Using human cancer cells and healthy skin cells that lack mitochondrial DNA, Sercel, Patananan et al. showed that cells that received mitochondria retained the new mitochondrial DNA. The technique uses readily accessible parts, meaning it can be performed quickly and inexpensively in any laboratory. It further only requires a small amount of donor starting material, meaning that even precious samples with limited material could be used as mitochondrial donors. This new technique has several important potential applications for mitochondrial DNA research. It could be used in the lab to create large numbers of cell lineswith known mutations in the mitochondrial DNA to establish new systems that test drugs or probe the interaction between mitochondrial and nuclear DNA. It could be used to study a broad spectrum of biological questions since mitochondrial function is essential for several processes required for life. Critically, it could also be used as a starting point to develop next-generation therapies capable of treating inherited mitochondrial genetic diseases in severely affected patients. Introduction Mitochondrial DNA (mtDNA) and nuclear DNA (nDNA) genome coordination regulates metabolism, epigenome modifications, and other processes vital for mammalian cell survival and activity (Patananan et al., 2018; Ryan and Hoogenraad, 2007; Singh et al., 2017). Together, these genomes encode >1100 mitochondrial proteins, with only 13 essential electron transport chain (ETC) proteins encoded within the mtDNA (Calvo and Mootha, 2010). The mitochondrial proteins encoded in the mtDNA and the nDNA must be compatible to support mitochondrial ETC activity. Mutations in mtDNA can impair the ETC by altering nDNA co-evolved ETC complex protein interactions, causing defective cellular respiration and debilitating diseases (Greaves et al., 2012). Furthermore, the coordination of these two genomes to transcribe, translate, and potentially modify appropriate levels of their respective gene products to maintain energetic and metabolic homeostasis is essential to the proper functioning of the ETC (Wolff et al., 2014). As a result, methods that enable pairing of specific mtDNA and nDNA genotypes in tractable systems are key to understanding the basic biology of mitonuclear interactions and their implications for health and disease. Our current inability to edit mtDNA sequences is a roadblock for many studies and potential applications. For example, endonucleases targeted to the mitochondrion inefficiently eliminate and cannot alter mtDNA sequences (Bacman et al., 2018). An exciting new bacterial cytidine deaminase toxin generates a limited repertoire of point mutations in the mtDNA; however, its efficiency remains low and it is unable to knock-in new gene sequences (Mok et al., 2020). Mitochondrial transfer between cells in vitro and in vivo provides a potential path forward for transplanting existing mtDNA sequences; however, the mechanisms controlling such transfers remain unknown (Dong et al., 2017; Torralba et al., 2016). Isolated mitochondrial transfer has been used to deliver mitochondria to a range of recipient cell types in vitro and even in vivo (Caicedo et al., 2015; Emani et al., 2017; Kitani et al., 2014); however, most studies using these methods observe only short-term changes to cell or organ performance and function. A small number of these studies have coincubated mitochondria with recipient cells and observed permanent retention of the exogenous mtDNA in mtDNA-deficient (so-called 'ρ0') cells using large doses of mitochondria or antibiotic selection schemes (Clark and Shay, 1982; Patel et al., 2017), although these approaches may not be possible when mitochondrial donor material is limited or does not possess a suitable selection marker. Methods that deliver mitochondria directly into ρ0 cells can increase stable mitochondrial transfer efficiency and employ a wider range of mitochondrial donor sources. Such methods include membrane disruption (King and Attardi, 1988; Wu et al., 2016) or fusion with enucleated cytoplasts (Wilkins et al., 2014). However, these methods are typically laborious, low-throughput, or depend on cancerous, immortal recipient cells lacking physiologic mitochondrial activity. An interesting recent study did report one desired mtDNA–nDNA clone and 11 false-positive clones using cybrid fusion with replication-limited cells, an achievement hampered by a low generation rate with unknown reproducibility or generalizability (Wong et al., 2017). There exist clinically relevant methods to replace the mtDNA of human cells, such as somatic cell nuclear transfer and pronuclear transfer that involve delivering nuclear genetic material from patients with mtDNA diseases into enucleated oocytes with non-mutant mtDNA genotypes (Hyslop et al., 2016; Tachibana et al., 2013). These methods hold potential for replacing deleterious mtDNA for the unborn, but they are technically challenging, low-throughput, dependent on high-quality patient samples, and prone to contamination by mutant mtDNA from the affected nuclear source material (Kang et al., 2016). Higher-throughput techniques that exchange non-native for resident mtDNAs in non-immortal somatic cells in tissue culture could enable studies of mtDNA–nDNA interactions and replace deleterious mtDNAs within cells with therapeutic potential (Patananan et al., 2016). Thus, a higher throughput, reproducible, and versatile mtDNA transfer approach to generate multiple desired 'stable isolated mitochondrial recipient' (SIMR) clones in replication-limited cells remains essential for statistically valid studies and potential translation of mitochondrial transplantation. Results MitoPunch mechanism uses fluid pressure to disrupt the plasma membrane We developed 'MitoPunch' as a simple, high-throughput mitochondrial transfer device consisting of a lower polydimethylsiloxane (PDMS) reservoir loaded with a suspension of isolated mitochondria, covered by a polyethylene terephthalate (PET) filter seeded with ~1 × 105 adherent cells (Figure 1A, Figure 1—figure supplement 1). MitoPunch uses a solenoid-activated plunger to transfer isolated mitochondria in a holding chamber by force into the cytosol of mammalian cells. Upon actuation, a mechanical plunger deforms the PDMS from below, which, as calculated by numerical simulation, generates pressure up to 28 kPa inside the PDMS chamber, propelling the suspension through numerous 3 µm pores in the PET filter. This pressure cuts the plasma membrane of recipient cells sitting atop the pores and delivers mitochondria into the cytoplasm of the cut cells (Figure 1B). To assess performance, we compared MitoPunch to mitochondrial coincubation (Kitani et al., 2014) and to MitoCeption (Caicedo et al., 2015), a method that uses centripetal force generated in a centrifuge to localize mitochondria to recipient mammalian cells (Figure 1C). In MitoCeption, a 1500 × g centripetal force draws isolated mitochondria to a recipient cell monolayer. We calculate that the suspended mitochondria exert a pressure of ~1.6 Pa on recipient cell membranes (Figure 1D) (see Materials and methods). Figure 1 with 1 supplement see all Download asset Open asset 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 Download elife-63102-fig1-data1-v2.zip Mitochondrial delivery into transformed and primary cells We isolated and delivered dsRed-labeled mitochondria from ~1.5 × 107 HEK293T cells (Miyata et al., 2014) into ~1 × 105 143BTK– ρ0 osteosarcoma cells and replication-limited BJ ρ0 foreskin fibroblasts in technical triplicate and measured the fraction of recipient cells positive for dsRed fluorescence by ImageStreamx MarkII imaging flow cytometry (Figure 2A). We define technical replication as independently performed mitochondrial deliveries using the same isolated mitochondrial preparation into recipient cells of the same passage. For 143BTK– ρ0 cells at ~2 hr post-delivery, imaging flow cytometry showed that MitoPunch yielded the lowest fraction of dsRed-positive cells compared to coincubation or MitoCeption. Similarly, for BJ ρ0 recipient cells, MitoPunch yielded the lowest fraction of dsRed-positive cells compared to coincubation or MitoCeption, although at lower levels relative to 143BTK– ρ0 recipients. This measurement assesses colocalization of mitochondria with recipient cells, and not necessarily the occurrence or mechanism of internalization of delivered mitochondria. These data suggest that the method of delivery and target cell type affect the efficiency of initiating mitochondria–recipient cell interactions. Figure 2 with 1 supplement see all Download asset Open asset 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. We quantified the number of discreet dsRed-spots in each cell ~2 hr following delivery from this data (George et al., 2004; Figure 2B and Figure 2—figure supplement 1). ImageStream spot count analysis of 143BTK– ρ0 recipient cells showed MitoPunch delivered a lower mean and median number of dsRed spots per cell than coincubation or MitoCeption. MitoPunch transfers into BJ ρ0 recipient cells yielded fewer mean spots/cell compared to coincubation and MitoCeption with an equivalent median number of spots/cell for MitoPunch and MitoCeption and slightly more for coincubation. Next, we used confocal microscopy to observe dsRed mitochondrial fluorescence in 143BTK– ρ0 recipients fixed 15 min post-transfer, which we chose for its robust mitochondrial acquisition (Figure 2C). We visualized mitochondrial localization with confocal microscopy by detecting dsRed protein from the donor mitochondria, shown in red, and labeling the recipient cell plasma membranes with either CellMask Green (coincubation and MitoCeption) or wheat germ agglutinin (MitoPunch), shown in green. Following MitoPunch, mitochondrial dsRed appeared to localize to pores in the filter insert and within the cytoplasm of cells, whereas coincubation and MitoCeption uniformly coated recipient cells with mitochondria, with greater mitochondrial association with recipient cells following MitoCeption. While all three methods initiate physical interactions between mitochondria and recipient cells, MitoPunch delivers mitochondria to the basal membranes of recipient cells at regions associated with the PET membrane pores, compared to a diffuse membrane association pattern seen with coincubation and MitoCeption. To investigate the capacity of these methods to disrupt recipient cell plasma membranes, we delivered the membrane impermeant dye propidium iodide (PI) by coincubation, MitoPunch, and MitoCeption to measure membrane disruption from delivery and quantified uptake by flow cytometry (Figure 2D; Novickij et al., 2017). Delivery into 143BTK– ρ0 cells by MitoPunch and MitoCeption resulted in similar percentages of PI-positive recipient cells, and both were greater than coincubation. Interestingly, BJ ρ0 cells showed comparable fractions of PI-positive cells to the 143BTK– ρ0 after coincubation and MitoCeption. However, MitoPunch yielded an approximately fivefold increase in the PI-positive fraction compared to all other conditions. These data show that MitoPunch and MitoCeption disrupt the plasma membranes of recipient cells for potential mitochondrial transfer, and the degree of disruption is cell type and delivery method dependent. Stable retention of transplanted mtDNA After verifying mitochondrial interaction with recipient cells by coincubation, MitoPunch, and MitoCeption, we next determined whether these methods result in permanent retention of exogenous mtDNA to generate SIMR cells. ρ0 cells cannot synthesize pyrimidines and therefore cannot proliferate or survive without supplemented uridine because of ETC impairment, so we used nucleotide-free medium prepared with dialyzed fetal bovine serum (SIMR selection medium) to select for SIMR cells with transplanted mtDNA and restored ETC activity (Grégoire et al., 1984; Figure 3A and B). BJ ρ0 cells survive longer under this selection scheme compared to the 143BTK– ρ0 (data not shown), so we included an additional selection phase by culturing these cells in nucleotide-free, glucose-free, galactose supplemented medium (galactose selection medium) (Robinson et al., 1992). We isolated and transferred HEK293T dsRed mitochondria into 143BTK– ρ0 and BJ ρ0 cells by coincubation, MitoPunch, and MitoCeption, performed SIMR selection in cell-type appropriate medium for 7 days, and quantified the number of viable clones by crystal violet staining (Figure 3C). Coincubation did not generate SIMR clones in 143BTK– ρ0 cells, in contrast to MitoPunch and MitoCeption, which each generated dozens of clones. BJ ρ0 cells with delivered HEK293T mitochondria by coincubation or MitoCeption did not form SIMR clones. MitoPunch generated numerous SIMR clones in both cell types, although fewer BJ ρ0 SIMR clones than in 143BTK– ρ0 cells, whereas MitoCeption only generated clones in 143BTK– ρ0 cells and was unable to form stable clones in replication-limited BJ cells. To assess the risk of mitochondrial donor cells surviving disruption during mitochondrial isolation and generating false positive SIMR clones, we performed three independent mitochondrial isolations, plated an aliquot from each isolation representing mitochondria isolated from ~1.5 × 107 HEK293T dsRed cells on 10 cm dishes, and carried these plates through the 10-day selection with SIMR selection medium before crystal violet staining for visual assessment (Figure 3—figure supplement 1). We observed no cell growth on any of the three plates, indicating a minimal incidence of donor cell survival through the mitochondrial isolation protocol. Figure 3 with 6 supplements see all Download asset Open asset 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. We next investigated whether differences in SIMR clone generation between 143BTK– ρ0 and BJ ρ0 cells were driven by sensitivity to differences in delivery pressure. We developed a MitoPunch device with adjustable plunger acceleration modulated by changing the circuit voltage (ImmunityBio). We generated independent voltage titration curves in 1 V increments (0 V – 5 V) for each cell type in technical triplicate at each voltage and used the same mitochondrial preparation for all samples for each cell type. All prior experiments in this study are controlled by delivering DPBS with calcium and magnesium to recipient cells by MitoPunch, but here we included a 0 V condition in which the seeded filter insert was positioned atop the PDMS reservoir and pressed against an aliquot of isolated mitochondrial suspension similar to deliveries with force, but without actuating the piston. We achieved maximum 143BTK– ρ0 SIMR clone generation with this tunable MitoPunch at 1 V, with a sharp reduction to background with increasing voltage (Figure 3D). The BJ ρ0 recipient also showed maximal SIMR generation at 1 V, with a shallow decline in SIMR generation efficiency to 5 V. Surprisingly, the 0 V condition consistently yielded a few SIMR clones in the 143 BTK– ρ0 recipients and inconsistently in the BJ ρ0 recipients. This result suggests that the pressure generated by sealing the filter insert against the PDMS reservoir is sufficient to generate SIMR clones at a low frequency. For all forthcoming MitoPunch trials we use the variable voltage MitoPunch device set to 1 V. We performed a similar force titration with MitoCeption by varying the maximum centripetal force, using a common mitochondrial preparation for all samples of both cell types. In 143BTK– ρ0 cells, we observed maximum clone generation at 1000 × g and 1500 × g, and we did not generate BJ ρ0 SIMR clones greater than the 0 × g background at any acceleration tested (Figure 3E). This background, present in both 143BTK– ρ0 and BJ ρ0 conditions at 0 × g, is likely from rare un-lysed donor cells from mitochondrial preparations directly pipetted into the culture medium of recipient cells during MitoCeption. We have infrequently observed imperfect donor cell lysis , usually in larger mitochondrial preparations, that results in rare, persistant dsRed fluorescent colonies as observed by fluorescence microscopy. True SIMR clones cannot produce the dsRed protein from donor mitochondria and lose fluorescence with time over these dsRed colonies maintain their fluorescence over the same (data not this donor cell background, MitoCeption yielded a response in SIMR clone generation from 143BTK– ρ0 recipients the MitoPunch deliveries into ρ0 cells et al., yielded maximal SIMR generation at a voltage than in the human cell showing that mitochondrial delivery pressure may be cell type dependent (Figure 3—figure supplement These data suggest that MitoPunch is to generate SIMR clones in replication-limited fibroblasts and SIMR generation efficiency on delivery pressure. We next quantified the reproducibility of mitochondrial preparation technique and the MitoPunch by triplicate MitoPunch transfers using three independent mitochondrial preparations from numbers of HEK293T dsRed biological (Figure 3—figure supplements 3 and We define biological replication here as mitochondrial preparations from independently of mitochondrial donor cells. Mitochondrial preparations and 3 as in Figure 3—figure supplement generated protein and each preparation yielded dozens of SIMR clones in all three technical MitoPunch deliveries with the exception of which resulted in two lower efficiency replicates. We quantified the number of SIMR clones generated per of mitochondrial loaded into the MitoPunch apparatus and observed a similar These results showed that mitochondrial isolation technique levels of isolated mitochondrial and that the MitoPunch technique yielded high numbers of SIMR clones. To enable desirable mtDNA–nDNA clone generation using limited starting material, such as mitochondria from rare cell we determined the minimal of mitochondrial required to generate SIMR clones. We performed coincubation, MitoPunch, and MitoCeption transfers into ~1 × 105 143BTK– ρ0 recipient cells using of dsRed mitochondria isolated from HEK293T cells and plated of the recipient cell on 10 cm We observed a similar between mitochondrial delivered and SIMR clones observed for MitoPunch and MitoCeption and mitochondrial protein suspended in of 1× pH transfer (Figure 3—figure supplement These results showed that although MitoPunch and MitoCeption generate SIMR clones from transformed recipient cells with similar efficiency per of mitochondrial the differences to the two of their relative the seeded PET filter from a dish to the MitoPunch apparatus resulted in medium being carried to the PDMS with the small of mitochondrial preparation delivered to the recipient cells, we observed that MitoPunch resulted in mitochondrial in the reservoir In the of mitochondrial material, we tested whether a used aliquot of isolated mitochondria can be to MitoPunch transfers to generate SIMR clones (Figure 3—figure supplement We performed 11 deliveries into 143BTK– ρ0 cells using one aliquot of mitochondrial and maximal SIMR clone generation from the and after which we observe a sharp reduction in SIMR cell and SIMR generation rate up to the transfer. These data showed that multiple MitoPunch transfers can be performed using a single aliquot of mitochondrial suspension when material is SIMR cells ρ0 mitochondrial respiration and we measured mitochondrial function in SIMR cells by the rate of and mitochondrial We isolated three independent 143BTK– ρ0 SIMR clones generated by MitoPunch or MitoCeption transfer of isolated HEK293T mitochondria and measured each rate using a mitochondrial test (Figure Figure supplement 1). To whether SIMR clone respiration stable through we the clones through two in uridine supplemented medium and measured cellular We that one MitoCeption clone its capacity and one MitoPunch clone was not viable after and (data not In the clones, basal and maximal and generation stable both We have performed numerous similar experiments using a range of recipients and mitochondrial and observed successful clone after (data not Figure with 2 supplements see all Download asset Open asset Mitochondrial DNA (mtDNA) ρ0 mitochondrial (A) rate quantification of basal and maximal and generation from two independent 143BTK– ρ0 HEK293T stable isolated mitochondrial recipient (SIMR) clones generated by MitoPunch and MitoCeption. data indicate clones that were and Error bars represent SD of technical replicates for SIMR cell measurements and for SIMR cell (B) microscopy of representative 143BTK– ρ0 HEK293T SIMR clones compared to 143BTK– HEK293T dsRed mitochondrial and 143BTK– ρ0 Mitochondria were stained with and labeled red, DNA was stained with and labeled green, and cell were stained with and labeled Scale bars indicate 15 µm. We the SIMR clones with and DNA to mitochondria and mtDNA by confocal microscopy (Figure and Figure supplement The MitoCeption clone that capacity showed a mitochondrial with no mtDNA (Figure supplement whereas the other SIMR clones generated by MitoPunch and MitoCeption mtDNA with mitochondrial These data show that the of 143BTK– ρ0 SIMR clones generated by either MitoPunch or MitoCeption have retained mtDNA, restored and mitochondrial Discussion of the mitochondrial genome is essential for the of mtDNA–nDNA interactions and for potential therapeutic applications of mitochondrial transfer. MitoPunch generates up to of SIMR clones in both transformed and recipient cells by a pressure sufficient to mammalian cell membranes in regions small to be within which cell