Journal Article

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

Shahrooz Rabizadeh, Alexander N Patananan, Garret W Guyot, Amy K Yu, Tianxing Man, Michael A Teitell, Alexander J Sercel, Pei-Yu Chiou, Ting-Hsiang Wu, Kayvan R Niazi

2021

Overview

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 simultaneousl y . 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. 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.

• Publisher: eLife Sciences Publications, Ltd,eLife Science Publications, Ltd,eLife Sciences Publications Ltd
• Subject: Animals / Biology (General) / Cell Biology / Cell Differentiation / Cell interactions / Cell Line / Cell Nucleus / Cell Nucleus - metabolism / Cells / Circular DNA / Cloning / DNA, Mitochondrial / DNA, Mitochondrial - genetics / Efficiency / Flow cytometry / Genetic research / Genomes / HEK293 Cells / Humans / Mammalian cells / Medicine / Mice / MitoCeption / Mitochondria / Mitochondria - metabolism / Mitochondrial DNA / mitochondrial transfer / mitochondrial transplantation / MitoPunch / Mutation / Oxidative phosphorylation / Phosphorylation / Plasma / Polyethylene terephthalate / Proteins / Q / QH301-705.5 / R / Science / stable isolated mitochondrial recipient / Tools and Resources / Transplantation