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The ethics and challenges on the future advances in xenogenic studies in the united states

Review Article Artificial Mitochondria Transfer: This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract The objective of this review is to outline existing artificial mitochondria transfer techniques and to describe the future steps necessary to develop new therapeutic applications in medicine.

  • In their model, they were able to transform around 30,000 recipient cells in just one procedure, making it a highly efficient method;
  • Both McCully and I-Rue Lai observed that the introduction of mitochondria to diseased ischemic tissue decreased damage and oxidative stress and improved recovery;
  • Given that mitochondrial donation involves the transfer of genetic but not nuclear material, this has led to uncertainty as to whether it should be regulated as egg or as tissue donation [ 155 ];
  • Having recently concluded this lengthy legalization process, the first babies conceived using MRT following this protocol are expected to be born this year in the UK;
  • During transport, mitochondria are enclosed and secured by membranes, thus protecting them from external damage.

The techniques currently in use today range from simple coincubations of isolated mitochondria and recipient cells to the use of physical approaches to induce integration. These methods mimic natural mitochondria transfer. In order to use mitochondrial transfer in medicine, we must answer key questions about how to replicate aspects of natural transport processes to improve current artificial transfer methods. Additionally, it is important that the field explores how artificial mitochondria transfer techniques can be used to treat different diseases and how to navigate the ethical issues in such procedures.

Without a doubt, mitochondria are more than mere cell power plants, as we continue to discover their potential to be used in medicine.

  • Additionally, each study verified the viability of the isolated mitochondria before injecting them into the given tissue using fluorescent probes dependent on membrane potential, including CMTMRos [ 107 ], JC1, and respirometry [ 49 ];
  • In response to this treatment, cytoplasmic vacuoles engulfed fragmented mitochondria and extruded them from apoptotic hepatocytes [ 81 ];
  • This indicates that mixing endogenous and transferred mitochondria across different species could potentially be restricted;
  • It has a double protective membrane and partial transcriptional independence from the nucleus, thereby making the mitochondria an item which can naturally be exchanged by microvesicles and nanotubes between cells [ 20 — 22 ];
  • Further assays need to be developed in vivo to fully understand the process of internalization related to AMT, possible heterogeneity across different tissues, and the effects of the transfer of mitochondria to harmed tissue.

Introduction Mitochondria are cell organelles descended from an alphaproteobacterial endosymbiont [ 1 ] and play a fundamental role in growth, differentiation, and survival beyond sustaining the energetics of the cell [ 23 ]. Diseases, tissue damage, and aging challenge the cell and its mitochondria, thereby affecting their integrity, function, and homeostasis [ 45 ].

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Cells naturally have the capacity to exchange intracellular material and especially mitochondria through different processes such as cell-to-cell contact, microvesicles, nanotubular structures, and other mechanisms [ 6 — 8 ].

Clark and Shay pioneered the artificial mitochondria transfer AMTwhich involved transferring mitochondria with antibiotic-resistant genes into sensitive cells, thereby enabling them to survive in a selective medium [ 9 ] and opening this new field of research.

Since the work of Clark and Shay, the process of artificial transfer has and continues to mimic aspects of naturally occurring cell transport, especially in the mechanisms cells naturally use to rescue other damaged cells. The AMT restores and increases respiration and proliferation and completes other cellular processes [ 510 — 16 ].

This review will consider key advances necessary to improve the current knowledge about the artificial transfer of mitochondria and how these techniques could be used therapeutically.

We will provide an overview of the features of the mitochondrial structure that are important in maintaining its integrity throughout artificial transfer [ 1314 ]. Next, we will discuss how a cell naturally protects the mitochondria during their transport by using intercellular bridges or microvesicles and the effects of the transferred mitochondria in the receiver cell [ 61718 ]. The in vivo artificial transfer of mitochondria was carried out at the same time as many in vitro assays [ 5712131619 ].

These approaches will be covered in the third section. For example, those assays performed by McCully in 2009 [ 16 ] and recently by Huang et al. The key to developing new lines of research in this field is determining the diseases in which AMT could be effective as well as the potential advantages of such therapeutic treatments over others. Taking this into account, it is essential that we further study the effectiveness of different donor sources of mitochondria in repairing recipient cells and determine how such findings can help to establish ethical guidelines that will facilitate future safety research and enable the development of new medical applications of AMT.

Without a doubt, more advances are needed to better understand and improve AMT and lay the foundation for its safe use in treating mitochondrial damage and related diseases. Structural and Functional Characteristics of Mitochondria for a Successful Artificial Transfer The mitochondrion is an organelle present in most of eukaryotic cells; it is in charge of ATP synthesis via oxidative phosphorylation OX-PHOScalcium metabolism, and the control of the apoptotic intrinsic pathway, among other functions.

At present, the mitochondrion is recognized as an endosymbiotic organism, whose noneukaryotic origin could facilitate its ability to be transferred from one cell to another. It has a double protective membrane and partial transcriptional independence from the nucleus, thereby making the mitochondria an item which can naturally be exchanged by microvesicles and nanotubes between cells [ 20 — 22 ]. Given that there is no cellular protection the ethics and challenges on the future advances in xenogenic studies in the united states performing AMT, it is important to conserve mitochondrial integrity after isolation when exposed to an extracellular environment.

The isolation procedure and stressors present outside the cell or organism like temperature change and surrounding media would greatly modified the structural stability, function, and potential effects of the mitochondria in the receiver cell [ 23 ].

Highlights

In this section, we will focus on key biological aspects that should be taken into consideration when the AMT to other cells is sought. The mitochondria evolved from a prokaryotic organism, and when it colonized the first protoeukaryotic cell, it developed a system of close communication with the nucleus by exchanging its own mtDNA sequences with it [ 2425 ].

It is estimated that mitochondria need almost 2000 proteins to work properly, but in many species, mtDNA encodes barely 63 proteins or less [ 2627 ] and most of these proteins are synthesized in the cytoplasm by means of ribosomes encoded in the nucleus and not by those of the mitochondria, thereby making them partially independent [ 28 ]. The interaction between nuclear and mitochondrial genes is essential for the organelle transcription, translation of proteins, and respiration [ 29 ].

Considering this close relationship, the compatibility between the mitochondria of one cell or species interacting with the nucleus of another could potentially affect their crosstalk, thereby inhibiting cell respiration and function [ 29 — 32 ]. These specific differences in the nuclear and mitochondria genome between cells or species could cause incompatibility if the auto, allo, and xenogenic AMT is pursued [ 13 ]. Its diameter varies between 0. Although its shape is defined as rounded or elongated, mitochondria can be very pleomorphic, or in other words, they may exhibit great morphological variations.

Some mitochondria could be fused and interconnected in networks, in contrast to the classic bean shape that appears in most illustrations [ 3536 ]. This organelle is characterized by a double lipoprotein membrane, each of which are about 7 nm thick.

The outer mitochondrial membrane is smooth, biochemically identical to the membranes of eukaryotic cells, and rich in cholesterol possibly contributing to the cell capacity to internalize this organelle when it is free in external medium [ 11 ]. The outer mitochondria membrane OMM serves as a barrier and a platform to exchange products between that cytoplasm and the intermembrane space [ 3738 ].

The OMM also protects the cell from any harmful product, like free radicals from the active metabolic processes carried out by the mitochondria [ 3739 ]. These factors can lead to the activation of proapoptotic multidomain Bcl-2 proteins, such as Bax or Bak [ 40 — 43 ].

A permeabilized or fragile OMM would not be effectively internalized after AMT by the receiver cell or even could activate apoptotic processes instead of repairing or increasing cellular functions [ 11 ]. Further studies should be completed in order to fully understand the interactions between the OMM and the receiver cell membrane and to understand the process of uptake.

The disruption of the IMM architecture could result in the alteration of the cristae dynamics in the mitochondria, consequentially affecting its capacity to fuse with other mitochondria and to produce ATP [ 374748 ]. One of the therapeutic possibilities of AMT is enabling the exchange of mtDNA from exogenous healthy mitochondria to damaged receiver mitochondria thereby contributing to the ATP production in which maintaining the integrity of IMM could favor the process.

Mitochondrial fitness is essential to maintain the integrity and functioning of the cell. Genetic variations in mitochondria and the presence of deleterious mutations in their DNA can alter their structure, function, and integrity. Many crucial aspects of their physiology are still not fully understood which are necessary to understand how physiological changes or stressors, like subproducts of the electron transport chain i.

In order to develop more efficient mechanisms and succeed the AMT, we must find ways to maintain their structural integrity during AMT, guaranteeing that the outer and inner membrane structures will be conserved and also that the mitochondria does not lose its function during the transfer, thus assuring the beneficial effects of the procedure [ 914 ].

Previous work about the AMT the ethics and challenges on the future advances in xenogenic studies in the united states mitochondrial function by fluorescent probes and electron microscopy being a key aspect of the transfer procedure [ 14164950 ]. There is still no information about the absolute or relative number of damaged versus healthy mitochondria during the AMT process. Obtaining this information could contribute to a better evaluation and comparison of the different AMT methods discussed in this review.

Cells and mitochondria change during the process of differentiation. It has been described that stem cell mitochondria are in a dormant and immature state: Through the process of differentiation and loss of their pluripotency, mitochondria proliferate and the quantity of DNA, the rate of respiration, and the generation of ATP synthase increase.

Artificial Mitochondria Transfer: Current Challenges, Advances, and Future Applications

These changes cause the mitochondria to develop an elongated morphology and swollen cristae. Its matrix also becomes more dense, being relocated to a wider extent in the cells [ 51 — 54 ]. It has not been studied whether the isolated mitochondria show variations on their effects on the recipient cells depending on the differentiation states as mitochondria show strong differences on their structure and metabolic profiles.

Questions like whether the cristae distribution change, ROS produced during the transfer, need to be answered and incorporated to the isolation and transfer protocols. In the next section, we will describe key aspects of natural intercellular mitochondria transfer, a cellular function which protects other cells from damage or stress [ 783355 ]. During transport, mitochondria are enclosed and secured by membranes, thus protecting them from external damage. In order to achieve successful artificial transfer, these mechanisms will need to be recreated in order to protect the mitochondria.

Natural Intercellular Mitochondrial Transfer To date, several groups have reported the horizontal transfer of mitochondria in different cell types in vitro and in vivo, describing a new cellular property [ 78215657 ]. Most of the work about mitochondrial delivery from one cell to another deals with the rescue of damaged cells by healthy ones, such mesenchymal stem cells hMSCs [ 85658 ].

Recently, this process was also observed occurring between astrocytes and neurons during focal cerebral ischemia [ 21 ].

Interestingly enough, in such cases, mitochondria from the retinal ganglion cell are transferred to astrocytes of the optic nerve head to be broken down and recycled [ 57 ]. From the first description of the transfer of intracellular material between cells in 2004 by Rustom et al. Considering the potential benefits of natural mitochondria transfer, there is great urgency to better comprehend, facilitate, and artificially replicate this process.

The transport of mitochondria from one cell to another is part of the dialogue necessary to the development and maintenance of homeostasis in multicellular organisms Figure 1 [ 59 ]. Mitochondria can travel from one cell to another by intercellular structures such as tunneling nanotubes TNTs and secreted cellular bodies, such as microvesicles [ 5203360 ].

In 2004, Rustom et al. Since then, a number of groups have studied the cells that produce TNTs and receive mitochondria and other intracellular cargo [ 563361 ]. Other reviews in this special issue and recently published work recapitulate the details of TNT structure generation, characteristics, and mitochondria transfer [ 6062 — 64 ]. Viable and nonfunctional mitochondria can be shared by the cell inducing different cellular responses from cellular rescue to promoting inflammation.

The first transfer mechanism is the microvesicle transport of mitochondria, it has been observed specially in MSCs in which the secreted microvesicles carrying mitochondria, once internalized by the recipient cells, induce its rescue from cellular damage and enhance the phagocytic properties of immune cells [ 182183 ].

The second way of transfer is by TNTs; many cells share the ability to produce them and transport mitochondria with proven effects in the rescue from cellular damage, metabolic reprogramming, and immune enhancement and it was also associated with its differentiation [ 65184 ].

During cellular stress, defective mitochondria can be released without being covered like in apoptotic or mitoapoptotic bodies and being naked promoting the immune response and inflammation [ 1777185 ]. TNTs are produced by the outgrowth of filopodia-like cell membrane protrusions that connect with the target cell. The membrane from each cell extends to fuse together, thereby forming a tightly connected bridge which is independent from any substrate [ 22 ].

TNTs contain a skeleton mainly composed of F-actin and transport proteins like MIRO1 that facilitate the active transfer of cargo and mitochondria along these structures [ 58 ].

TNTs were first described in rat-cultured pheochromocytoma PC12 cells [ 6 ], and subsequent studies have shown that they connect a wide variety of cell types. These studies provide more evidence that TNTs are involved in mitochondrial transport between cells, the repair of cell damage, the activation of enhanced immune responses, and cell metabolic reprogramming [ 58336165 ]. The directionality of the transport of intracellular material and mitochondria through the TNTs is not fully understood.

It is important to define what factors promote the donation of material and their effects on the recipient cells. Stressed hippocampal neurons and astrocytes initiated the formation of TNTs after p53 activation. This signaling pathway triggered caspase 3, which decreased S100A4 in injured cells and caused cells with a high level of S100A4 to become receptor cells [ 66 ].

By these results, the authors proposed that damaged cells need to transfer cellular contents to healthy ones, in a process related to the spread of danger signals but no insights about mitochondrial participation were given. In contrast, Spees et al. Bidirectional transport of mitochondria is also plausible as it was observed between malignant mesothelioma cells. These cells produce more TNTs than normal mesothelioma cells, but interestingly, their proliferation was inversely correlated with TNT formation during their culture in low serum, hyperglycemic, acidic growth medium [ 69 ].

These represent just a few examples of the extensive literature about the exchange of intracellular material and mitochondria and its directionality. Yet, mitochondrial transport is not fully understood. For example, the field still needs to define the cell types that produce TNTs and deliver cargo to recipient cells [ 68 ]. The determination of the directionality of and conditions necessary for mitochondria transfer between different cells is essential to understanding the potential role of this process in helping cells exposed to stress or during the transmission of danger warning signals among cells.

Another important question that still remains unanswered is the the ethics and challenges on the future advances in xenogenic studies in the united states why MSCs have a greater propensity to form TNTs compared with other cells. Many groups of researchers that use lung disease models have corroborated that mitochondria can be transferred to other cells in vivo. The delivery of mitochondria into injured cells increased ATP levels which in turn maintained cellular bioenergetics and recovered epithelium functions.

A follow-up study in lung disease models rotenone-induced lung injury and allergen-induced asthma contributed to the understanding of the mechanisms involved in mitochondrial transfer through nanotubes, confirmed the protective effect of mitochondrial donation, and revealed a Miro1-regulated mitochondrial movement from MSC to damaged recipient epithelial lung cells [ 58 ].