Here, we present a protocol including mitochondrial tracing, direct co-culture procedures of mesenchymal stem cells (MSCs) and retinal pigment epithelial cells (ARPE19), as well as the methods for observing and statistically analyzing tunneling nanotubes (TNT) formation and mitochondrial transfer to characterize mitochondrial exchange via TNTs between MSCs and ARPE19 cells.
Mitochondrial transfer is a normal physiological phenomenon that occurs widely among various types of cells. In the study to date, the most important pathway for mitochondrial transport is through tunneling nanotubes (TNTs). There have been many studies reporting that mesenchymal stem cells (MSCs) can transfer mitochondria to other cells by TNTs. However, few studies have demonstrated the phenomenon of bidirectional mitochondrial transfer. Here, our protocol describes an experimental approach to study the phenomenon of mitochondrial transfer between MSCs and retinal pigment epithelial cells in vitro by two mitochondrial tracing methods.
We co-cultured mito-GFP-transfected MSCs with mito-RFP-transfected ARPE19 cells (a retinal pigment epithelial cell line) for 24 h. Then, all cells were stained with phalloidin and imaged by confocal microscopy. We observed mitochondria with green fluorescence in ARPE19 cells and mitochondria with red fluorescence in MSCs, indicating that bidirectional mitochondrial transfer occurs between MSCs and ARPE19 cells. This phenomenon suggests that mitochondrial transport is a normal physiological phenomenon that also occurs between MSCs and ARPE19 cells, and mitochondrial transfer from MSCs to ARPE19 cells occurs much more frequently than vice versa. Our results indicate that MSCs can transfer mitochondria into retinal pigment epithelium, and similarly predict that MSCs can fulfill their therapeutic potential through mitochondrial transport in the retinal pigment epithelium in the future. Additionally, mitochondrial transfer from ARPE19 cells to MSCs remains to be further explored.
Mitochondria serve as the primary energy source for most cell types, with mitochondrial dysfunction particularly impacting high-energy-demanding tissues like the retina1. Metabolic alterations in the retina can trigger a bioenergetic crisis, ultimately resulting in the death of photoreceptors and/or RPE cells2. Mesenchymal stem cell (MSC)-based therapies have demonstrated efficacy in treating ocular degeneration, and one of the precise mechanisms underlying the beneficial effects of MSCs on retinal tissues may be attributed to functional mitochondrial transfer3,4,5,6. In 2004, Rustom et al. first reported the phenomenon of mitochondrial transfer through a novel cell-to-cell interaction facilitated by tunneling nanotubes (TNTs)7.
In 2D culture, tunneling nanotubes (TNTs) are identified by their thin (20-700 nm) membrane protrusions ranging from tens to hundreds of nanometers in length, which are suspended over the substrate and can directly establish connections between two or more homotypic and heterotypic cells. These structures are notably enriched in F-actin and facilitate the transport of cargo, such as mitochondria, between cells. Additionally, TNTs possess openings at both ends, enabling the continuity of cytoplasmic content between interconnected cells8.
It is difficult to detect TNT-mediated mitochondrial transfer in vivo due to the dense cellular arrangement and challenges in tracking mitochondria. In vitro experimentation, utilizing cell co-culture and mitochondrial tracing techniques, allows for the observation of TNT formation and mitochondrial transfer8,9. We also observed the phenomenon of TNT-mediated mitochondrial transfer by co-culturing MSCs and retinal pigment epithelial cells in vitro10.
Many previous studies have only observed unidirectional mitochondrial transfer from MSCs to other cells3,4,5,6. Previously, we also tried to analyze the bidirectional mitochondrial transfer using two kinds of cells labeled with mito-tracker green and mito-tracker red, respectively, but the crosstalk of the dyes interfered with the experimental results. To study mitochondrial bidirectional transfer more precisely, here, we constructed two cell lines with different mitochondrial fluorescence using the lentiviral transfection technique, and subsequently, observed and analyzed the phenomena of TNT formation and mitochondrial bidirectional transfer by direct co-culture in vitro.
In brief, a step-by-step and actionable protocol is described here as to how to trace mitochondria, co-culture MSCs with ARPE19 cells, and analyze TNT formation and mitochondrial transfer. The results of this experiment demonstrated TNT-mediated bidirectional mitochondrial transfer, which not only proved that mitochondrial transport is a common physiological phenomenon but also showed the potential therapeutic ability of MSCs on retinal cells.
1. Generation of MSC-mito-GFP and ARPE19-mito-RFP cell lines
2. Direct co-culture of MSC and ARPE19 cells
NOTE: In this co-culture system, MSC-mito-GFP cells will serve as the donor cells while ARPE19-mito-RFP cells will function as the recipient cells. To distinguish between donor and recipient cells, we traced recipient cells.
3. Indirect co-culture of MSC and ARPE19 cells in a transwell system
4. Cytoskeleton staining
NOTE: Protect from light throughout the experiment.
5. Confocal imaging
NOTE: Confocal imaging is performed according to the operation manual and may vary between microscopes. Here we give only some of the key steps.
6. Data analysis
The schematic diagram illustrating the direct co-culture of mesenchymal stem cells (MSC) and ARPE19 cells is depicted in Figure 1. MSCs, engineered to express mito-GFP, as the donor cells and ARPE19-mito-RFP cells with violet-labeled cytoplasmic membranes as recipient cells were co-cultured at a ratio of 1:1. Following a 24 h co-culture period, the cells were stained for phalloidin and examined using confocal microscopy. The resulting cell populations included MSC-mito-GFP cells, ARPE19-mito-RFP cells, ARPE19-mito-RFP cells containing mito-GFP, and MSC-mito-GFP cells containing mito-RFP. Notably, the presence of mito-GFP in ARPE19 cells and mito-RFP in MSCs suggested mitochondrial bidirectional transfer.
Specific details of TNT formation are presented in Figure 2. These structures can establish intercellular connections between cells of the same type (Figure 2A-C), as well as between cells of different types (Figure 2D). Tunneling nanotubes (TNTs) serve as conduits connecting adjacent cells, exhibiting a distinctive F-actin-rich membrane structure with open ends facilitating cytoplasmic continuity and the transport of mitochondria. Utilizing Z-axis imaging, it is evident that TNTs are not firmly anchored to the extracellular matrix but rather suspended within the culture medium, thereby distinguishing them from conventional cellular protrusions. Mitochondrial transfer is evident in Figure 3, where violet-positive ARPE19 cells exhibit dispersed, green-dotted fluorescence indicative of mitochondria originating from MSCs (Figure 3A). Violet-negative MSCs containing red-dotted fluorescence represent the mitochondrial transfer from ARPE19 cells (Figure 3B). Figure 4 shows TNT formation and mitochondrial transfer under super-resolution imaging, from which we can see more details of mitochondrial transfer via TNT. Video 1 shows the dynamics of mitochondrial transport through TNT.
To further demonstrate that the prerequisite for mitochondrial transfer is physical contact, such as TNT, rather than mitochondrial secretion and uptake, we used a previously reported co-culture method10. As shown in Figure 5A, MSCs and ARPE19 cells were seeded in the upper and lower chambers of a transwell plate, respectively. They were separated by a filter with 0.4 µm pores, which blocked the direct contact between MSCs and ARPE19 cells but allowed the passage of mitochondria. After 24 h of co-culture, ARPE19 cells in the lower chamber were assayed and little mito-GFP was observed from MSCs in the upper chamber (Figure 5B). These results suggest that mitochondrial transfer requires direct contact between MSCs and ARPE19 cells.
Ultimately, the quantification of TNT formation and mitochondrial transfer was conducted, with the corresponding results depicted in Figure 6. MSCs were significantly more capable of donating mitochondria than ARPE19 cells (p = 0.0286). These findings indicate that MSCs possess the capability to transfer mitochondria to retinal pigment epithelial cells.
Figure 1: Schematic view of co-culture of MSCs and ARPE19 cells. MSCs expressing mito-GFP and ARPE19-mito-RFP cells labeled with CellTrace Violet are co-cultured for 24 h at a ratio of 1:1. Mito-GFP in violet+ cells marks the transfer of mitochondria from MSCs to ARPE19 cells, while Mito-RFP in violet– cells marks the transfer of mitochondria from ARPE19 cells to MSCs. Abbreviations: GFP = green fluorescent protein; RFP = red fluorescent protein; MSCs = mesenchymal stem cells. Please click here to view a larger version of this figure.
Figure 2: TNT formation between MSCs and ARPE19 cells. White arrowheads indicate TNT formation, green arrowheads indicate mitochondria with mito-GFP and red arrowheads indicate mitochondria with mito-RFP. (A,B) TNT formation between ARPE19 cells. (C) TNT formation between MSCs. (D) TNT formation between MSCs and ARPE19 cells. Z-axis imaging showing TNTs suspended in culture medium. Scale bars = 25 µm. Abbreviations: MSCs = mesenchymal stem cells; TNTs = tunneling nanotubes. Please click here to view a larger version of this figure.
Figure 3: Mitochondrial transfer from MSCs to ARPE19 cells. (A) The white dashed circle indicates mitochondrial (mito-GFP) transfer from MSCs to ARPE19 cells, and (B) the yellow dashed circle indicates mitochondrial (mito-RFP) transfer from ARPE19 cells to MSCs. Scale bars = 20 µm. Abbreviation: GFP = green fluorescent protein; RFP = red fluorescent protein; MSCs = mesenchymal stem cells. Please click here to view a larger version of this figure.
Figure 4: TNT formation and mitochondrial transfer between MSCs and ARPE19 cells by super-resolution microscopy. (A and B) Mitochondrial transport from MSCs to ARPE19 cells. (C) Detail of TNTs under super-resolution imaging. (D) Green mitochondria from MSCs present in ARPE19 cells under super-resolution imaging. Scale bar = 5 µm. Abbreviations: MSCs = mesenchymal stem cells; TNTs = tunneling nanotubes. Please click here to view a larger version of this figure.
Figure 5: Almost undetectable mitochondrial transfer in indirect co-culture in transwell system of MSCs and ARPE19 cells. (A) Schematic view of co-culture of MSCs and ARPE19 cells in transwell system. (B) Representative images of ARPE19 cells in the bottom of the transwell plate after indirect co-culture with MSCs for 24 h. Please click here to view a larger version of this figure.
Figure 6: Quantification of TNT formation and mitochondrial transfer. (A) TNT formation, (B) mitochondrial transfer. Abbreviations: M = MSCs = mesenchymal stem cells; A = ARPE19 cells; TNTs = tunneling nanotubes; M-A = TNT formation between MSCs and ARPE19 cells; M-M = TNT formation between MSCs; A-A = TNT formation between ARPE19 cells. n = 4, mean ± SD, Mann-Whitney U-test, *p < 0.05. Please click here to view a larger version of this figure.
Supplemental Figure S1: Mapping of plasmid pCT-Mito-copGFP. Mito-GFP (CYTO102-PA-1) was purchased. The Mitochondria Cyto-Tracer, pCT-Mito-GFP (CMV), fuses with copGFP via a Cox8 tag, resulting in the labeling of mitochondria with copGFP. This copGFP fusion is driven by the CMV promoter, enabling robust expression in cell lines such as HeLa, HEK293, and HT1080. Mito-RFP was modified from mito-GFP by replacing GFP with RFP; it carries a Puromycin resistance gene. Lentiviral packaging of this plasmid is done by specialized companies. Please click here to download this File.
Video 1: TNT-mediated mitochondrial transport between MSCs and ARPE19 cells. TNT is generated through the bidirectional movement of MSC and ARPE19 cells, facilitating the transfer of mitochondria from MSC cells to ARPE19 cells. Abbreviations: MSCs = mesenchymal stem cells; TNTs = tunneling nanotubes. Please click here to download this Video.
Numerous studies have demonstrated that the phenomenon of TNT-mediated mitochondrial transfer is a prevalent physiological process in various types of tissue cells10,11,12,13. Functional mitochondrial donation from MSCs to cells with mitochondrial dysfunction exhibits strong therapeutic potential3,14,15,16,17,18.
Here, to present TNT-mediated bidirectional mitochondrial transfer between MSCs and ARPE19 cells, we constructed MSC-mito-GFP and ARPE19-mito-RFP cell lines. Next, the two kinds of cells were co-cultured for 24 h, and cytoskeletal staining was performed at the end of the co-culture. By confocal imaging, we observed and counted TNT formation and mitochondrial transfer rate. The outcomes of these observations may be influenced by factors such as the proportion of co-cultured cells and cell density. During co-culture, it is imperative to thoroughly mix the two cell types and maintain them in a single-cell state to minimize the formation of cell colonies by homotypic cells, thereby reducing the occurrence of mitochondrial transfer between the two cell types.
Flow cytometric analysis can similarly be used to analyze mitochondrial transfer rates, and this method is simpler than postimaging statistics. However, the mitochondrial transfer rate calculated using this method will be somewhat lower than the actual value, because small amounts of mitochondrial transfer may not be recorded. In this protocol, the counting of imaging results needs to be done very carefully to avoid omissions or incorrect counting of fluorescent spurious signals.
The study of bidirectional mitochondrial transfer by constructing two cell lines with mitochondrial fluorescence is an innovation of this protocol. The method avoids the interference of fluorescent dyes and makes the experimental results more credible. The transfer of mitochondria from retinal cells to MSCs deserves further study, for example, to explore changes in the proportion of mitochondria transferred in both directions under stressful conditions.
By adhering to the experimental procedures outlined in this protocol, we were able to ascertain that MSCs can transfer mitochondria to retinal pigment epithelial cells through tunneling nanotubes. This phenomenon may serve as a plausible mechanism underlying the advantageous impacts of MSCs on retinal tissues, thereby aiding in the elucidation of MSCs-driven regenerative therapies for ocular tissues.
The authors have nothing to disclose.
We thank Guangzhou CSR Biotech Co. Ltd for imaging with their commercial super-resolution microscope (HIS-SIM), data acquisition, SR image reconstruction, analysis, and discussion. This work is partly supported by the National Natural Science Foundation of China (82125007,92368206) and the Beijing Natural Science Foundation (Z200014).
0.25% Trypsin-EDTA | Gibco | 25200-056 | |
4% paraformaldehyde | Solarbio | P1110 | |
6-well plate | NEST | 703001 | |
15 mL centrifuge tube | BD Falcon | 352097 | |
24-well plate | NEST | 702001 | |
ARPE19 cells | ATCC | CRL-2302 | Cell lines |
Bovine serum albumin (BSA) | Beyotime | ST025 | |
CellTrace violet | Invitrogen | C34557 | |
Cover slide | NEST | 801007 | |
DMSO | sigma | D2650 | |
DPBS | Gibco | C141905005BT | |
DMEM/F-12-GlutaMAX | Gibco | 10565-042 | |
Fetal Bovine Serum (FBS) | VivaCell | C04002-500 | |
FluorSave Reagent | Millipore | 345789 | |
MSCs | Nuwacell | RC02003 | Cell lines |
ncMission | Shownin | RP02010 | |
Pen Strep | Gibco | 15140-122 | |
pCT-Mito-GFP | SBI | CYTO102-PA-1 | Plasmid; From https://www.systembio.com/mitochondria-cyto-tracer-pct-mito-gfp-cmv |
Puromycin | MCE | HY-B1743A | |
Pipette | Axygen | TF-1000-R-S | |
Phalloidin | Invitrogen | A22287 | |
Triton X-100 | Solarbio | T8200 | |
Transwell plate | Corning | 3470 |
.