1. Introduction

With 600,000 deaths in the United States and 18 million overall, cardiovascular diseases represent the primary cause of death worldwide ^[1][2]. In addition to their enormous health, ethical and financial issues, they are expected to increase in the coming years especially due to the rise of aging individuals worldwide, their increasingly extending life span and the occurrence of concomitant age-related cardiovascular changes ^[3]. One reason for the disturbing high mortality rate is the circumstance that cardiac tissue is not able to regenerate diseased cells properly since the yearly tissue turnover rate is only around 1%. Insults like ischemia caused by myocardial infarction and subsequent reperfusion or cardiomyopathies therefore lead to substantial impairments of cardiac function. Traditional treatments with pharmaceutical and exogenous substrate interventions (statins, beta-adrenergic blockers, angiotensin converting enzyme inhibitors, aspirin, clopidogrel etc.) and/or surgeries provide often only limited success with a remaining negative impact on post ischemic recovery and cell viability ^[4]. Up to date reliable treatments against cardiac damage and for cardiac rejuvenation are urgently needed and under extensive investigation ^[5].

In the past decades the field of stem cell based treatment strategies emerged as a promising approach to prevent or tackle consequences of disease-caused cardiovascular changes in various application areas. By aiming to rescue damaged cells and regenerate damaged tissue or modulate inflammatory pathways extensive research has been conducted over the last decades mainly with mixed or negative results. Hurdles like the optimal application strategy, poor survival and engraftment of the cells as well as a lack of proliferation and the danger of rejection as well as the need for immunosuppressive agents presented drawbacks in the use of cell-based treatment approaches ^[6][7][8][9]. In addition recent research implicated paracrine factors of the stem cells and the microenvironment to enhance the benefit compared to direct stem cell applications used ^{[10][11][12][13]}. Based on these findings so called “stem-less” approaches emerged as interesting alternatives to the use of stem cells in cardiac medicine. Various positive effects have been described and were attributed to the beneficial effects such as improvement of the microenvironment, the removal of senescent cells, the optimization and application of exosomes, microRNAs (miRNAs) and latest also mitochondria ^[14][15][16]. Especially the latter might represent an interesting therapeutic target: Cardiomyocytes heavily rely on an adequate energy metabolism and mitochondria constitute the main energy source of cells. Mitochondrial changes have been repeatedly found in cardiovascular alterations and targeting them yielded improvements in function and regeneration. In recent years mitochondrial transplantation therefore emerged as an interesting and so far, promising therapeutic option in a variety of diseases, including cardiovascular.

However despite the reported positive effects, previous in vivo studies used mainly autologous derived mitochondria. With regard to the probable patient cohort- old and diseased individuals- and the observable aging effects of various cell types and mitochondria themselves this might pose obstacles in the application of this approach. The questions arise if mitochondria from somatic tissues of the same individual are safe, effective and expedient in these cases or if other cell sources like, for example, stem cells or mitochondria derived from younger individuals would exert a more beneficial effect.

2. Using Stem Cells for Mitochondrial Transplantation

Mitochondria for in vivo cardiovascular applications are usually derived from autologous muscle biopsies and produce significant therapeutic effects. But due to demographic changes and the increased occurrence of cardiovascular diseases the patients relevant for these therapeutic strategies are usually older than the currently used (young) animal models in which these strategies have been tested. In addition inherited mitochondrial diseases would prohibit the usage of autologous mitochondria. This poses the question if the traditionally used muscle biopsies are optimal for this approach and if not other cell sources or strategies could be options and used as donors for the applied mitochondria.

Although autologous derived mitochondria from the skeletal muscle are currently the most common procedure for mitochondrial transplantation in the field of cardiovascular research, other sources of mitochondria have also been successful used: Transplantation of autologous mitochondria from heart tissues and liver tissues as well as allogeneic and syngeneic derived mitochondria have been described. Especially in the field of neuroscience, stem cells are common sources. The principal proof that mitochondria can be transferred into homogeneic and xenogeneic cells has been shown by various groups: Kitani et al. co-incubated these cells successfully with isolated mitochondria ^[17]. Stem cell derived mitochondria have been used for transplantation in cancer cells [154]. Islam et al. showed the successful mitochondrial transfer from human and mouse BMSCs to mice alveolar epithelium by forming gap junction channels and mitochondria-containing microvesicles leading to the presence of human mtDNA and an increase of ATP content in the alveolar tissue. When HeLa cell-derived mitochondria were used for transplantation in rabbit cardiomyocytes a co localization at the cell surface within 2h followed by an internalization in the myocytes within 8h was seen. However, in this study, the majority of mitochondria remained in the extracellular space and just 3 - 7% of mitochondria seemed to be incorporated ^[18].

The fact that mitochondria provoke lower or no immunogenicity compared to cell-based approaches and that they are significantly smaller thereby reducing the risk for cell-induced vessel occlusion, transplantation options from different individuals or species may be worth investigating ^[19].

Interestingly, an increased mitochondrial transfer efficacy was observed for iPS-derived MSCs than for BM MSCs when MSCs were co cultured with rat airway epithelial cells and in vivo ^[20]. In a mouse model of anthracycline-induced cardiomyopathy iPSC-MSCs displayed a higher human mitochondrial retention rate compared to BM-MSCs ^[21]. This might be due to a high expression of MIRO1 and TNFαIP2 which made iPSC-MSCs “more responsive to TNFα-induced tunneling nanotube (TNT) formation for mitochondrial transfer to CMs, which is regulated via the TNFα/NFκB/TNFαIP2 signaling pathway” ^[21].

Using healthy non-autologous mitochondria might even be more beneficial than using autologous mitochondria. For example mitochondria in diabetic patients and rodent models have been shown to be dysfunctional, having reduced respiratory capacity and ATP production rate ^[22]. In addition, autologous mitochondria might not be an option in patients with mitochondrial myopathies and mutational changes in the mtDNA ^[23].

Although already applied (often investigated in subgroups of the experimental studies; see section “Mitochondrial transplantation for heart diseases”), the usage of stem cell derived mitochondria for in vivo purposes is currently under-investigated and important questions for practical considerations, like identifying the best cell source, isolation protocols, improvements of mitochondria quantities and preservation of these mitochondria are worth to be further examined. Traditionally HSCs were thought to be mitochondria sparse but recent studies report on abundant mitochondria: Almeida showed a high mitochondrial mass and high mtDNA content in HSCs. mtDNA content declined with differentiation of the HSCs. But baseline oxygen consumption, mitochondrial ATP production and maximal respiratory capacity were lower in HSCs compared to MMPs and CPs; turn over capacity of mitochondria was low in HSCs.

The importance of identifying the best cell source has been recently evidenced by an investigation from Paliwal and colleagues. In their study SCs from dental pulp or Wharton jelly showed better efficacy for transfer than other stem cells. The authors concluded that a careful selection on cell source should be made since MSCs with higher mitochondrial bioenergetics display higher rescue potential with lower mitochondrial transfer ^[24].

Another aspect which should be regarded is the fact, that mitochondria or better said the cell type and tissue differs in metabolic profile, mitochondrial content and energy sources and demands using glucose, fatty acids, ketone bodies or lactate as source. In the healthy heart, mainly (>95%) fatty oxide oxidation occurs, which switches to glycolytic pathways in the failing heart ^[25]. A direct comparison between brain, heart, liver, skeletal muscle and kidney of Wistar rats revealed hearts having a 5–10 fold higher cytochrome C oxidase activity than the other organs with liver being the lowest. However, the CoX/CS ratio was the highest in liver and kidney. mtDNA and mitochondrial content per cell were the highest in cardiac muscle, followed by skeletal muscle ^[26]. However, in vivo studies using different mitochondrial sources seemed to display no functional differences ^[27], which was confirmed by investigations of different muscle sources for in vitro mitochondrial transfer into cardiomyocytes ^[28].

The subpopulations of mitochondria, SSM vs IMF, have been shown to differ in regard of morphology and functionality ^[29]. These two subpopulations seem to exhibit different age-related metabolic alterations: whereas SSMs are mainly unaltered IMF exhibit alterations in oxidative phosphorylation ^[30]. However, no functional differences were observed in transplantation experiments ^[4]. Studies indicate however different susceptibilities of mitochondrial subtypes to diseases, which should be taken into consideration when treatments for affected patients are considered.

3. Conclusions

Despite previous promising usage of mitochondria for transplantations important considerations regarding significant aging effects of somatic cells, stem cells and mitochondria as well as factors like safety issues, tissue sources and possible disease effects deserve further investigations when mitochondrial transplantations are to be used for future applications. Factors influencing stem cell and mitochondria function include age of the cells, probably previous divisions of the cells, heterogeneity of stem cells as well as mitochondria and likely tissue source and additional diseases. Furthermore after the first positive reports, the time of treatment for the most beneficial effect and repetitions of applications should be further investigated: positive effects have been shown pre ischemia, prior to and during reperfusion as well as after delayed application. The quantity of mitochondria seems to be less critical as only a small number of mitochondria is needed for improving cardiac functions. The development of further safety and storage solutions for mitochondria could improve applications. Following the first promising reports of stem cell derived mitochondria further research especially considering the differences of autologous (maybe collection in early life stages and asservation for later use) vs. allogeneic vs. syngeneic sources deserve further investigations and will surely lead to exiting new developments during the upcoming years.