May the force be with you: Transfer of healthy mitochondria from stem cells to stroke cells

Abstract

Stroke is a major cause of death and disability in the United States and around the world with limited therapeutic option. Here, we discuss the critical role of mitochondria in stem cell-mediated rescue of stroke brain by highlighting the concept that deleting the mitochondria from stem cells abolishes the cells' regenerative potency. The application of innovative approaches entailing generation of mitochondria-voided stem cells as well as pharmacological inhibition of mitochondrial function may elucidate the mechanism underlying transfer of healthy mitochondria to ischemic cells, thereby providing key insights in the pathology and treatment of stroke and other brain disorders plagued with mitochondrial dysfunctions.

Keywords: Cerebral ischemia; cellular bioenergetics; energy metabolism; inflammation; regenerative medicine; transplantation.

Figure 1.

Characterization of mitochondrial transfer in stroke. (A) Our overarching hypothesis advances the concept that mitochondrial dysfunction is a major secondary cell death process in ischemic stroke, which is accompanied by impaired cellular bioenergetics. A key downstream pathway of dysfunctional mitochondria involves inflammation that compromises the neurovascular unit (neurons, astrocytes, and endothelial cells). Mitochondria-based regenerative medicine via stem cell therapy, combined with tissue-plasminogen activator (tPA) recanalization that restores blood flow and re-entry of nutrients and energy to the cerebral vasculature, is hypothesized to sequester inflammation and to aid in recapturing CNS homeostasis. We propose mitochondria repair as a novel stroke therapeutic pathway applicable to regenerative medicine; in particular, our envisioned mechanism of action entails functional restoration of bioenergetics through the transfer of healthy mitochondria into the ischemic brain, harnessing a therapeutic vasculome that robustly sequesters the toxic inflammation-plagued secondary cell death associated with stroke and other disorders characterized by mitochondrial deficits. (B) Mitochondrial morphology and distribution in normal and OGD-exposed cortical neurons. Representative pictures show cortical neurons stained for the neuronal marker MAP2 (red) and mitochondrial ATP5A (green). Scale bar = 10 µm. Cortical neurons (2 × 10⁵) were plated and allowed to stabilize for five days. Cells were then exposed to control conditions (complete media, 5% CO₂, 21% O₂) or OGD (PBS, 5% CO₂, 95% N₂) for 90 min. Twenty-four hours later, cells were probed with MAP2 and ATP5A antibodies. Boxed areas are shown as magnified images of tubular and circular mitochondria. (C) Quantification of healthy tubular mitochondria in control and OGD neurons. Neurons exposed to OGD exhibited ∼35% less tubular mitochondria than controls (***p < 0.0001 by Student’s t test). (D) Calcein assay showing significantly reduced cell survival (∼55%; *p < 0.05) upon OGD exposure (b) relative to controls (a). Bar graphs represent mean ± SD of three individual experiments. Scale bar = 100 µM. (E) Bright-field images of neurons grown in ambient conditions, OGD, and OGD co-cultured with EPCs. Arrows indicate outgrowth processes, which were significantly reduced in OGD conditions and rescued when neurons were co-cultured with EPCs. (F) Oxygen uptake in digitonin-permeabilized neurons (2 × 10⁶) was recorded with a Clark’s oxygen electrode (Oxygraph Plus System, Hansatech, UK) under phosphorylating conditions as described. The activity of citrate synthase was evaluated by spectrophotometry (EnSpire plate reader, Perkin Elmer, USA) as described. Data are shown as mean ± SD. Statistical analysis was performed with one-way ANOVA followed by Bonferroni’s post-hoc test. (G) Oxygen consumption in mitochondrial-enriched fractions from naive and stroke animals. Adult Sprague-Dawley rats were exposed to stroke via the middle cerebral artery occlusion. After 4.5 h, animals received stereotaxic transplants of either human EPCs (4 × 10⁶ viable cells in 9 µl sterile saline) or vehicle (9 µl sterile saline), delivered into the cortex corresponding to the anticipated penumbra area. At 72 h post-transplantation, animals were euthanized, and the cortical region corresponding to the transplant site was harvested. Enriched mitochondrial fractions were obtained by mechanical tissue disruption and differential centrifugation. Mitochondrial bioenergetics was assessed using the Seahorse XFe96 analyzer. Real-time measurements (mean ± SD, n = 6 samples per condition) of OCR (upper panel) and ECAR (lower panel) were recorded in the presence of ADP (40 mM), followed by the addition of oligomycin (25 µg/ml), FCCP (40 µM), and rotenone (2 µM)/antimycin A (40 µM) injected sequentially as shown. OCR and ECAR of mitochondria from stroke + vehicle and stroke + EPCs illustrate the difference in metabolic profiles between the two conditions. Data are shown as mean ± SD. Asterisks represent significant differences between the two groups (*p < 0.01) computed by one-way ANOVA followed by Bonferroni’s post-hoc test. (H) Co-localization of EPCs’ mitochondria in stroke rats’ neurons. Sprague Dawley male rats (∼250 g) were subjected to 60 min MCAO, and 3 h later, EPCs (0.4 × 10⁶ cells in 3 µl) were stained with MitoTracker Deep Red FM (Invitrogen) at 500 nM and then transplanted into the ischemic penumbra. At day 1 after transplantation, the animals were sacrificed, perfused, and brains were harvested, cryosectioned and processed for NeuN (Abcam) IHC staining. Confocal images were captured at 60 × and 180 × (Green: NeuN; Red: EPCs’ mitochondria; Blue: DAPI). Results revealed that mitochondria transfer occurs in vivo from EPCs to neurons. The transfer of mitochondria can be seen in few cells adjacent to the original implantation site (a, magnified in a′), while higher number of neuronal cells with EPC mitochondria (white arrows) are more apparent within the transplant site (b). (I) Mitochondrial mass in EPCs and EPC-derived Rho0 cells. EPCs were cultured in MEM alpha (Gibco) supplemented with 20% FBS, 1%NEAA, 1% GlutaMax and 1% Penicillin/Streptomycin. Rho0 cells were obtained by treating EPCs with 0.1 µM ethidium bromide and supplemented with 1 mM pyruvate and 0.2 mM uridine for three weeks. Cells were stained with 500 nM MitoTracker Deep Red FM (Life Technologies), fixed and probed with an anti-β-tubulin antibody (Abcam, Cambridge, MA). Confocal images were captured at 180 × . Blue: DAPI; red: β-tubulin; cyan: mitochondria. Mitochondrial DNA copy number was evaluated by qRT-PCR. Total DNA (gDNA and mtDNA) was prepared by using Nucleospin tissue kit (TaKaRa, Cat. #740952.50) starting from Rho0 EPCs (4 × 10⁶ cells) and EPCs (6 × 10⁶ cells). Rho0 cells showed less than 1% mtDNA copy number relative to EPCs. ATP5A: ATP synthase, alpha subunit; ECAR: Extra Cellular Acidification Rate; FCCP: carbonilcyanide p-triflouromethoxyphenylhydrazone; MAP2: microtubule associated protein 2; MCAO: Middle Cerebral Artery Occlusion; mtDNA: mitochondrial DNA; OCR: Oxygen Consumption Rate; qRT-PCR: Real-Time Quantitative Reverse Transcription PCR; RCR_ADP: Respiratory Control Ratio under coupling conditions in the presence of 1 mM ADP.