bims-mitrat 2025-02-02 papers

Sci Rep. 2025 Jan 27. 15(1): 3350

The role of homogenization cycles and Poloxamer 188 on the quality of mitochondria isolated for use in mitochondrial transplantation therapy.

Ryosuke Takegawa, Kei Hayashida, Atsushi Murao, Yusuke Endo, Cyrus E Kuschner, Jacob Kazmi, Eriko Nakamura, Ping Wang, Lance B Becker.

Mitochondrial transplantation (MTx) offers a promising therapeutic approach to mitigate mitochondrial dysfunction in conditions such as ischemia-reperfusion (IR) injury. The quality and viability of donor mitochondria are critical to MTx success, necessitating the optimization of isolation protocols. This study aimed to assess a rapid mitochondrial isolation method, examine the relationship between mitochondrial size and membrane potential, and evaluate the potential benefits of Poloxamer 188 (P-188) in improving mitochondrial quality during the isolation process. Mitochondria were isolated from pectoral muscle biopsies of adult male Sprague-Dawley rats using an automated homogenizer. MitoTracker Deep Red (MTDR) staining and flow cytometry were used to assess mitochondrial purity, while the JC-1 assay evaluated membrane potential. Mitochondrial size groups were compared for membrane potential differences. Homogenization frequency and P-188 supplementation (1 mM) were assessed for their effects on mitochondrial membrane potential and particle size, and particle counts. The rapid isolation method yielded mitochondria that retained sufficient membrane potential to be effectively inhibited by carbonyl cyanide 3-chlorophenylhydrazone (CCCP), a disruptor of mitochondrial membrane potential. Larger mitochondria exhibited significantly higher JC-1 ratios, indicating greater membrane potential. Excessive homogenization (10 cycles) reduced membrane potential compared to 3 cycles homogenization (P = 0.026). P-188 significantly increased the JC-1 ratio from 10.26 ± 2.57 to 33.78 ± 17.78 (P = 0.023). Particle size and count analysis revealed that 10 cycles homogenization significantly increased particle count compared to 3 cycles homogenization (P = 0.0001), but was associated with smaller particle sizes (P = 0.0031). The rapid mitochondrial isolation method produced viable mitochondria, with larger mitochondria exhibiting superior membrane potential. Reducing homogenization frequency and incorporating P-188 improved mitochondrial quality and preserved particle size. These strategies offer promising strategies for optimizing MTx protocols. Further refinement of these techniques is necessary for their clinical application in MTx therapy.

Keywords: JC-1; Mitochondrial isolation; Mitochondrial membrane potential; Mitochondrial transplantation; Poloxamer 188

DOI: https://doi.org/10.1038/s41598-025-86760-y

Adv Sci (Weinh). 2025 Jan 31. e2412621

Osteoblast-Derived Mitochondria Formulated with Cationic Liposome Guide Mesenchymal Stem Cells into Osteogenic Differentiation.

Hye-Ryoung Kim, Seonjeong Woo, Hui Bang Cho, Sujeong Lee, Chae Won Cho, Ji-In Park, Seulki Youn, Gyuwon So, Sumin Kang, Sohyun Hwang, Hye Jin Kim, Keun-Hong Park.

While mitochondria are known to be essential for intracellular energy production and overall function, emerging evidence highlights their role in influencing cell behavior through mitochondrial transfer. This phenomenon provides a potential basis for the development of treatment strategies for tissue damage and degeneration. This study aims to evaluate whether mitochondria isolated from osteoblasts can promote osteogenic differentiation in mesenchymal stem cells (MSCs). Mitochondria from MSCs, which primarily utilize glycolysis, are compared with those from MG63 cells, which depend on oxidative phosphorylation. Mitochondria from both cell types are then encapsulated in cationic liposomes and transferred to MSCs, and their impact on differentiation is assessed. Mitochondria delivery from MG63 cells to MSCs grown in both two- and three-dimensional cultures results in increased expression of osteogenic markers, including Runt-related transcription factor 2, Osterix, and Osteopontin, and upregulation of genes involved in Bone morphogenetic protein 2 signaling and calcium import. This is accompanied by increased calcium influx and regulated by the Wnt/β-catenin signaling pathway. Transplantation of spheroids containing MSCs with MG63-derived mitochondria in bone defect animal models improves bone regeneration. The results suggest that delivery of MG63-derived mitochondria effectively guides MSCs toward osteogenesis, paving the way for the development of mitochondria-transplantation therapies.

Keywords: MSCs; delivery; liposome; mitochondria; mitochondrial transfer; osteogenic differentiation

DOI: https://doi.org/10.1002/advs.202412621

Life Med. 2024 Apr;3(2): lnae017

Mitochondrial transplantation for the treatment of cardiac and noncardiac diseases: mechanisms, prospective, and challenges.

Xinyi Wang, Zhiyuan Liu, Ling Zhang, Guangyu Hu, Ling Tao, Fuyang Zhang.

Mitochondrial transplantation (MT) is a promising therapeutic strategy that involves introducing healthy mitochondria into damaged tissues to restore cellular function. This approach has shown promise in treating cardiac diseases, such as ischemia-reperfusion injury, myocardial infarction, and heart failure, where mitochondrial dysfunction plays a crucial role. Transplanting healthy mitochondria into affected cardiac tissue has resulted in improved cardiac function, reduced infract size, and enhanced cell survival in preclinical studies. Beyond cardiac applications, MT is also being explored for its potential to address various noncardiac diseases, including stroke, infertility, and genetic mitochondrial disorders. Ongoing research focused on refining techniques for mitochondrial isolation, preservation, and targeted delivery is bolstering the prospects of MT as a clinical therapy. As the scientific community gains a deeper understanding of mitochondrial dynamics and pathology, the development of MT as a clinical therapy holds significant promise. This review provides an overview of recent research on MT and discusses the methodologies involved, including sources, isolation, delivery, internalization, and distribution of mitochondria. Additionally, it explores the effects of MT and potential mechanisms in cardiac diseases, as well as non-cardiac diseases. Future prospects for MT are also discussed.

Keywords: cardiac diseases; heart; mitochondria; mitochondrial transplantation; noncardiac diseases

DOI: https://doi.org/10.1093/lifemedi/lnae017

Iran J Basic Med Sci. 2025 ;28(1): 38-48

Effects of combo therapy with coenzyme Q10 and mitochondrial transplantation on myocardial ischemia/reperfusion-induced arrhythmias in aged rats.

Soleyman Bafadam, Behnaz Mokhtari, Alireza Alihemmati, Reza Badalzadeh.

Objectives: Ischemia/reperfusion (IR)-induced ventricular arrhythmia, which mainly occurs after the opening of coronary artery occlusion, poses a clinical problem. This study aims to investigate the effectiveness of pretreatment with coenzyme Q10 (CoQ10) in combination with mitochondrial transplantation on IR-induced ventricular arrhythmias in aged rats.
Materials and Methods: Myocardial IR induction was performed by left anterior descending coronary artery occlusion for 30 min, followed by re-opening for 24 hr. CoQ10 was administered intraperitoneally at a dosage of 10 mg/kg/day for two weeks before inducing IR. At the start of reperfusion, 500 µl of the respiration buffer containing 6×106±5×105 mitochondria/ml of respiration buffer harvested from the pectorals major muscle of young donor rats were injected intramyocardially. To investigate arrhythmias, the heart's electrical activity during ischemia and the first 30 min of reperfusion were recorded by electrocardiogram. After 24 hr of reperfusion, cardiac histopathological changes, creatine kinase-MB, nitric oxide metabolites (NOx), oxidative stress markers (malondialdehyde, total anti-oxidant, superoxide dismutase, and glutathione peroxidase), and the expression of genes regulating mitochondrial fission/fusion were measured.
Results: Pretreatment with CoQ10 in combination with mitochondrial transplantation reduced ventricular arrhythmias, cardiac histopathological changes, and creatine kinase-MB levels. Simultaneously, this combined therapeutic approach increased myocardial NOx levels, fostering an improved oxidative balance. It also triggered the down-regulation of mitochondrial fission genes, coupled with the up-regulation of mitochondrial fusion genes.
Conclusion: The combination of CoQ10 and mitochondrial transplantation demonstrated a notable anti-arrhythmic effect by elevating NOx levels, reducing oxidative stress, and improving mitochondrial fission/fusion in aged rats with myocardial IRI.

Keywords: Arrhythmia; Coenzyme Q 10 Mitochondria; Myocardial ischemia/reperfusion injury; Oxidative stress

DOI: https://doi.org/10.22038/ijbms.2024.80092.17348