Mitochondrial Transplantation: Could Photobiomodulation Play a Role?

Mitochondrial transplantation has recently become a hot topic in biomedical research. The number of articles published each year has grown from hardly any before 2015 to >40 articles in each of the past 3 years.

The concept actually dates from a landmark article published by Clark and Shay in Nature in 1982. ¹ These authors isolated mitochondria from cells, which had developed resistance to cytotoxic antibiotics, and when these mitochondria were incubated with antibiotic-sensitive cells, the resistance was transferred, suggesting that the mitochondria had been taken up by the recipient cells by a process of endocytosis. This process could be linked to the theoretical evolutionary origin of mitochondria, which has been attributed to prehistoric bacteria that were engulfed by primitive cells during the “great oxygenation event” 2.4 billion years ago, resulting in the eventual production of eukaryotic cells. ²

Nowadays it has been shown that mitochondria isolated in the laboratory from a variety of donor cells can be transferred into recipient cells. This result has been demonstrated by fluorescent labeling of the mitochondria before transfer and subsequent observation by microscopy, and by the restoration of respiration in rho-zero cells that had been engineered to be devoid of mitochondria. ³ Many of the in vitro studies have employed direct microinjection of isolated mitochondria into the recipient cells.

This approach originated from in vitro fertilization research where oocytes from a mother with a mitochondrial disease were microinjected with mitochondria from a healthy donor followed by fertilization with the father's sperm. Although this approach led to birth of a healthy baby, it was later replaced by the transfer of the nucleus from the unhealthy oocyte, into the cytoplasm of a denucleated healthy donor oocyte followed by fertilization. Nevertheless, these so-called “three parent babies” remain controversial, and there is concern about the possibility of “carry over” of unhealthy mitochondria into the recipient oocyte along with the nucleus. ⁴

In order for mitochondrial transplantation to be useful as a clinical treatment, the isolated mitochondria must be taken up by the recipient cells by some kind of endocytosis, because microinjection would be unfeasible on a large scale. The exact mechanism of endocytosis involved has turned out to be quite controversial, because different recipient cell types may have different propensities to take up exogenous isolated mitochondria. One study investigated mitochondrial internalization into cardiomyocytes using various inhibitors to block different types of endocytosis (micro-pinocytosis, macro-pinocytosis, clathrin-dependent endocytosis, and tunneling nanotubes). ⁵ Because only cytochalasin D inhibited uptake they concluded that actin-dependent endocytosis was the mechanism of uptake, but did not explain how the mitochondria escaped from the endosomes once they were internalized.

It has been found that cells with defective mitochondria take up exogenous mitochondria more readily than healthy cells. The beneficial effects include higher adenosine triphosphate (ATP) production, lower generation of reactive oxygen species, and stimulation of mitochondrial biogenesis. The beneficial effects lasted in cells in vitro for at least 53 cell doublings. However, one caveat has been raised, because fragmented extracellular mitochondria could act as damage-associated molecular patterns and trigger immune responses in the recipient.

The use of mitochondrial transplantation as a treatment for a wide variety of tissue damage and degenerative organ disorders has generally involved the isolation of mitochondria from a source tissue or cell preparation, rapidly followed by injection into the host organ either directly (32-gauge needle) or through the arterial blood supply. ⁶ The isolation procedure can use autologous tissues such as skeletal muscle or liver, autologous cells such as platelets, or cell lines such as various types of stem cells. The isolation procedure must be rapid (∼90 min) because mitochondria degrade rapidly, and care should be taken to use the optimum buffers to preserve respiratory competence. Some authors have proposed that mitochondria encapsulated inside extracellular vesicles would perform better than free mitochondria. One procedure was described that used differential filtration to isolate 10 billion functional mitochondria from 0.1 g of muscle biopsy in only 30 min, and claimed it could be used intraoperatively or at the patient bedside. ⁷

Many of the research applications of mitochondrial transplantation have focused on the heart, the brain, or the spinal cord. In the heart it has been found to increase coronary blood flow and enhance ventricular function after a heart attack or ischemia reperfusion injury. ⁶ In the brain it has been tested in acute ischemic stroke, traumatic brain injury, Alzheimer's disease, and Parkinson's disease. ⁸

A pioneering controlled clinical trial was carried out in 24 pediatric patients with cardiogenic shock caused by ischemia-reperfusion injury occurring during surgery. In the 10 patients who received mitochondrial transplantation, their recovery was quicker (2 vs. 9 days for controls) and with fewer cardiovascular events. ⁹ At present there are six clinical trials of mitochondrial transplantation for a variety of indications (heart, brain, and others) recorded on ClinicalTrials.gov (NCT04998357, NCT03639506, NCT02851758, NCT05669144, NCT03384420, and NCT04976140).

Because photobiomodulation (PBM) is known to principally act on mitochondria, it is reasonable to ask whether PBM could play a role in mitochondrial transplantation. It is well known that PBM can increase membrane potential, respiration, oxygen consumption, and ATP production when applied to isolated mitochondria in vitro. It would be possible to apply PBM to the mitochondria during or immediately after isolation to improve their function, which is critical in the success of the procedure. Another possibility is to shine the light on the recipient tissue after the mitochondrial injection.

So far there has been one report that investigated this approach in a rat model of spinal cord injury (SCI). ¹⁰ Zhu et al. first isolated mitochondria from platelets in 10 mL of rat blood, and then co-incubated them with rat dorsal root ganglion cells in vitro, with or without PBM using an 810 nm laser. The combined treatment increased uptake of fluorescent mitochondria into the neurons, and increased axonal growth. These effects could be abrogated by treatment with a specific inhibitor of connexin 36, called 18β glycyrrhetic acid. Connexin 36 is a protein involved in formation of pores and gap junctions in neurons.

In a rat model of SCI caused by compression of the spinal cord, mitochondria (300,000 in 10 μL) were stereotactically injected into the lesion, followed by implantation of a fiber optic and surgical closure. This allowed 810 nm laser light to be delivered for 60 min/day over 14 days. Rats receiving the combination of mitochondria plus PBM showed significant improvements in motor performance, along with increased mitochondrial function and less oxidative stress compared with either treatment alone. They concluded that PBM increased the expression of connexin 36 in the neurons, thus improving the uptake of the mitochondria into the host cells.

Considering the beneficial effects of PBM, not only on the mitochondria themselves, but also on the host recipient cells and tissue, further studies of this novel combination approach would appear to be warranted.