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).

1. Generation of MSC-mito-GFP and ARPE19-mito-RFP cell lines
<ol>
	<li>Cell culture 
	NOTE: Only MSCs are used here as an example.
	<ol>
		<li>Culture human MSCs in a 6-well plate in hMSC medium with 1% penicillin and streptomycin (see Table of Materials) until the cell density reaches 80%-90%. Depending on the density of cell growth, change the medium once every 2-3 days.
		<ol>
			<li>Remove the original medium and wash the cells once with phosphate-buffered saline (DPBS) (see Table of Materials). Add 1 mL of the trypsin-EDTA-DPBS mixture (0.05% Trypsin-EDTA: 0.25% Trypsin-EDTA diluted with DPBS at a ratio of 1:5; see Table of Materials), and incubate for 3-4 min (depending on the cells) in a 37 &#176;C incubator. 
			NOTE: Observe cell digestion under a microscope and stop digestion if most of the cells are rounded and detached.</li>
			<li>Gently tap the 6-well plate to detach the cells adhered to the surface, and then add 1 mL of fresh complete medium to terminate the digestion.</li>
			<li>Gently resuspend all the cells with a pipette gun and subsequently transfer all collected cells into a 15 mL centrifuge tube.</li>
			<li>Centrifuge the cells at 200 &#215; g for 5 min.</li>
			<li>Aspirate the supernatant and resuspend the cells by adding 1 mL of fresh medium. Take 10 &#181;L of cell suspension for cell counting.</li>
			<li>Eighteen to 24 h before lentiviral transfection, seed MSC cells at a density of 1 &#215; 105 cells per well in 6-well plates, ensuring that the cell density at the time of transfection is approximately 50%.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Transfection of lentivirus 
	NOTE: All operations related to lentiviral transfection need to be performed in a biosafety cabinet.
	<ol>
		<li>Replace the initial medium with 2 mL of fresh medium the next day, and add 25 &#181;L of lentivirus suspension (5.00E+08 TU/mL) with adjuvants, followed by incubation at 37 &#176;C. 
		NOTE: Lentivirus packaging was performed using the Mito-GFP (pCT-Mito-copGFP) plasmid (see Table of Materials and Supplemental Figure S1) by a company.</li>
		<li>After 6 h of incubation, replace the virus-containing medium with 2 mL of fresh medium. Continue to incubate and change the medium once a day. 
		NOTE: Significant mitochondrial fluorescence expression is observed in MSCs 48 h post transfection, with further enhancement evident at the 72 h time point.</li>
		<li>Screen successfully transfected cells by supplementing the culture medium with puromycin (1 &#181;g/mL) 2 days post transfection. Concurrently, add an equivalent concentration of puromycin to mesenchymal stem cells lacking viral transfection as a control. 
		NOTE: The mito-GFP viral vector is engineered to include the puromycin resistance gene.</li>
		<li>Change the medium daily with the introduction of puromycin (1 &#181;g/mL). Stop puromycin addition upon near-complete cell death in the control group. 
		NOTE: The medium does not contain this concentration of puromycin. The addition of puromycin is required only when the medium is added to a 6-well plate.</li>
		<li>Continue to culture without puromycin until cells are passaged and frozen. Name this type of cell as MSC-mito-GFP. 
		NOTE: ARPE19 cells were derived from ATCC Cell Bank and cultured in DMEM/F12 containing 10% FBS and 1% penicillin and streptomycin (see Table of Materials), and the methods of their digestion and passage are the same as that of MSCs. Plasmid Mito-RFP was modified from Mito-GFP by a company. Finally, we named the successfully transfected cells as ARPE19-mito-RFP.</li>
	</ol>
	</li>
</ol>
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.
<ol>
	<li>Label ARPE19-mito-RFP cells with CellTrace Violet (see Table of Materials) in the cytoplasmic membrane. 
	NOTE: The excitation and emission of dye CellTrace violet are 405 nm and 450 nm, respectively.
	<ol>
		<li>Prepare a CellTrace Violet (see Table of Materials) stock solution by adding 20 &#181;L of dimethyl sulfoxide (DMSO) to a vial of CellTrace Violet Reagent and mixing well immediately prior to use.</li>
		<li>Grow the ARPE19-mito-RFP cells to the desired density of 80%-90%.</li>
		<li>Dilute the CellTrace Violet stock solution in prewarmed (37 &#176;C) DPBS to the desired working concentration (1:1,000). This is the loading solution. 
		NOTE: The dilution of CellTrace Violet must be performed with DPBS and cannot be replaced with culture medium as this will lead to very poor staining results.</li>
		<li>Remove the culture medium and replace it with 1 mL of loading solution. Incubate the cells for 10 min at 37 &#176;C. 
		NOTE: If conditions permit, the staining can be observed under a fluorescence microscope after 5 min. Staining can be terminated as soon as the desired results are achieved, as some cells may not be able to tolerate prolonged exposure to DPBS.</li>
		<li>Remove the loading solution, wash the cells 2x with DPBS, and replace with 2 mL of fresh complete medium. Incubate the cells for at least 10 min after staining to allow the CellTrace Violet to undergo acetate hydrolysis. 
		NOTE: This step is necessary for the cells to return to their optimal state and to avoid interfering with subsequent experiments.</li>
	</ol>
	</li>
	<li>Prepare a 24-well plate by adding 50 &#181;L of DPBS into each well. Subsequently, insert the cover glass (with a specified diameter of 15 mm) (see Table of Materials) into the plate and allow it to settle for 1 min. Remove the DPBS from the wells, utilizing negative pressure to ensure the cover slide is close to the bottom of the dish.</li>
	<li>Trypsinize donor MSCs and recipient ARPE19 cells as described in step 1.1.1.</li>
	<li>Cell counting
	<ol>
		<li>Place 10 &#181;L of cell suspension on a counting plate for enumeration. 
		NOTE: If the density of cells is too high, they can be diluted 10-fold with medium and then proceed as above.</li>
		<li>Seed 10,000 MSCs and 10,000 ARPE19 cells in 500 &#181;L of medium per well of a 24-well plate. Mix the cells well before adding them to the 24-well plate. See the calculations shown below given an MSC count of A/mL and an ARPE19 cell count of B/mL. 
		NOTE: For 4 wells, 40,000 MSCs and 40,000 ARPE19 cells are required in 2 mL of medium. 
		Volume of MSC suspension (C) containing 40,000 MSCs = 40,000/A &#181;L 
		Volume of ARPE19 suspension (D) containing 40,000 MSCs = 40,000/B &#181;L 
		Make up the volume to 2 mL with 2 - (C + D) mL of medium.</li>
	</ol>
	</li>
	<li>Observe the cells under a microscope, shake the plate until the cells are evenly distributed, and then incubate them in the culture for 24 h.</li>
</ol>
3. Indirect co-culture of MSC and ARPE19 cells in a transwell system
<ol>
	<li>Perform cell culture, labeling, digestion, and counting as described in steps 2.1 to 2.4.1.</li>
	<li>Seed 20,000 ARPE19 cells in 1 mL of medium per well of a 24-well plate. 
	NOTE: In this experiment, we used ARPE19 cells without labeled mitochondria.</li>
	<li>Place the insert into the well where the cell has been seeded.</li>
	<li>Seed 10,000 MSCs in 100 &#181;L of medium in the upper cell chamber per well.</li>
	<li>Repeat step 2.5.</li>
</ol>
4. Cytoskeleton staining
NOTE: Protect from light throughout the experiment.
<ol>
	<li>Remove the medium from the 24-well plates. Wash all cells once with 500 &#181;L/well of 4% polyformaldehyde (PFA), add 500 &#181;L of fresh 4% PFA, and fix the samples for 20 min. 
	NOTE: The insets of the transwell system are discarded. 
	NOTE: Because nanotubes are very fragile, we washed the cells directly with 4% PFA instead of DPBS to maximize the retention of intact nanotubes.</li>
	<li>Remove the fixative and wash the samples for 3 x 10 min with DPBS.</li>
	<li>Add 500 &#181;L/well of blocking solution composed of 0.5% Triton X-100 (see Table of Materials) and 4% bovine serum albumin (BSA) (see Table of Materials) and incubate for 1 h at room temperature.</li>
	<li>Remove the blocking solution and wash the samples for 3 x 10 min with DPBS.</li>
	<li>Preparation of phalloidin staining solution (see Table of Materials) 
	NOTE: The excitation and emission of phalloidin are 650 nm and 668 nm, respectively.
	<ol>
		<li>Prepare stock solutions by dissolving the vial contents in 150 &#181;L of anhydrous DMSO to yield a 400x stock solution.</li>
		<li>Prepare staining solution by diluting the 400x storage solution to 1x with DPBS. This is the phalloidin staining solution.</li>
	</ol>
	</li>
	<li>Add 200 &#181;L of phalloidin staining solution to each well and incubate for 1 h at room temperature.</li>
	<li>Recover the staining solution and wash the sample 3 x 10 min with DPBS. 
	NOTE: When washing the cells with DPBS, it will be better to leave the plate on a shaker.</li>
	<li>Pick out the cover glass with a syringe needle and allow it to air dry. 
	NOTE: The optimal state is one in which no visible liquid can be seen on the cover glass but it is also not dry.</li>
	<li>Apply a small amount of mounting medium (see Table of Materials) onto the slide and carefully flip the cover glass on&#160;the slide to ensure that the medium evenly coats the entire surface. Seal the edges with nail polish. 
	NOTE: Wait for a few minutes to allow the nail polish to dry out.</li>
	<li>Capture a confocal image promptly or transfer it to a slice box and store it at 4&#160;&#176;C for a brief duration. 
	NOTE: Ideal storage time is 2 weeks. If this time is exceeded, the imaging effect may deteriorate.</li>
</ol>
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.
<ol>
	<li>Start the confocal microscope and open four laser channels (405, 488, 561, 640) according to the operation manual.</li>
	<li>Access the software on the computer and perform preliminary parameterizations of the software.
	<ol>
		<li>Add the fluorescent channels CF-405, CF-488, CF-561 and CF-640 within the Process Management panel.</li>
		<li>Adjust the exposure time of each fluorescence channel to 200 ms and set the gain intensity to 2 in the Camera Control panel.</li>
		<li>Adjust the laser/LED combiner to a value of 80.</li>
	</ol>
	</li>
	<li>Manipulate the external control panel of the microscope to adjust the objective lens to a magnification of 20x and ensure that the lens is positioned at its lowest setting.</li>
	<li>Position the slide in an inverted orientation on the carrier table.</li>
	<li>Activate the 405 nm laser and manipulate the focus using the coarse and fine focusing spirals. Observe the sample under the eyepiece until blue fluorescent cells are visible.</li>
	<li>Select Live Imaging in the software interface on the computer, switch to the CF-405 channel, and readjust the fine focus helix until a distinct image of the cells exhibiting blue fluorescence is visible on the computer screen.</li>
	<li>Adjust the objective lens to 40x magnification and transition to the CF-640 channel, then carefully adjust the focus until a distinct cellular microfilament structure is visible.</li>
	<li>Maintaining a constant focal length, transition to the CF-405, CF-488, and CF-561 channels sequentially, and modify the parameters of optimal exposure time, gain intensity, and laser/LED combiner for imaging within each channel, as necessary.</li>
	<li>Activate the Z Image Stack function and define the beginning and ending points of the Z-axis by manipulating the focal length to capture an image with depth using the Z-axis.</li>
	<li>Click the Start button in the Process Management panel to take a photo.</li>
	<li>Select 10 fields of view randomly for each slice. 
	NOTE: There should be no overlap between any two fields of view.</li>
	<li>Activate the time-lapse function, specifying a total imaging duration of 24 h and a photo interval of 5 min following identification of the desired field of view. 
	NOTE: To capture a time-lapse sequence, it is necessary to inoculate cells in a glass-bottomed dish and position them within a ventilated box containing 5% CO2 gas.</li>
	<li>Save and export all images. 
	NOTE: To enhance the scrutiny of TNT and mitochondrial transfer, super-resolution imaging was employed. Super-resolution imaging was performed using commercialized HIS-SIM, termed HIS-SIM (High Intelligent and Sensitive SIM) provided by a company. Images were acquired using a 100x/1.5 NA oil immersion objective.</li>
</ol>
6. Data analysis
<ol>
	<li>For the statistical analysis of each image, quantify the total number of cells, the number of nanotubes, the number of Violet-positive ARPE19-mito-RFE cells, the number of Violet-positive cells exhibiting green fluorescence (representing the number of ARPE19 cells with mitochondrial transfer from MSCs), and the number of Violet-negative cells exhibiting red fluorescence (representing the number of MSC cells with mitochondrial transfer from ARPE19 cells).
	<ol>
		<li>To quantify TNT formation, calculate the ratio of the number of intercellular nanotubes to the total number of cells.</li>
		<li>Calculate the mitochondrial transfer rate as the ratio of the number of Violet-positive cells exhibiting green fluorescence to the total number of Violet-positive cells (representing the mitochondrial transfer from MSCs to ARPE19 cells) or the ratio of the number of Violet-negative cells exhibiting red fluorescence to the total number of Violet-negative cells (representing the mitochondrial transfer from ARPE19 cells to MSCs).</li>
	</ol>
	</li>
</ol>

Investigating Mitochondrial Transfer in MSC and ARPE19 Co-Cultures

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The authors declare that they possess no conflicting interests.

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</sup...

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.

<table><tbody><tr><td>0.25% Trypsin-EDTA</td><td>Gibco</td><td>25200-056</td><td></td></tr><tr><td>4% paraformaldehyde</td><td>Solarbio</td><td>P1110</td><td></td></tr><tr><td>6-well plate</td><td>NEST</td><td>703001</td><td></td></tr><tr><td>15 mL centrifuge tube</td><td>BD Falcon</td><td>352097</td><td></td></tr><tr><td>24-well plate</td><td>NEST</td><td>702001</td><td></td></tr><tr><td>ARPE19 cells</td><td>ATCC</td><td>CRL-2302</td><td>Cell lines</td></tr><tr><td>Bovine serum albumin (BSA)</td><td>Beyotime</td><td>ST025</td><td></td></tr><tr><td>CellTrace violet</td><td>Invitrogen</td><td>C34557</td><td></td></tr><tr><td>Cover slide</td><td>NEST</td><td>801007</td><td></td></tr><tr><td>DMSO</td><td>sigma</td><td>D2650</td><td></td></tr><tr><td>DPBS</td><td>Gibco</td><td>C141905005BT</td><td></td></tr><tr><td>DMEM/F-12-GlutaMAX</td><td>Gibco</td><td>10565-042</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>VivaCell</td><td>C04002-500</td><td></td></tr><tr><td>FluorSave Reagent</td><td>Millipore</td><td>345789</td><td></td></tr><tr><td>MSCs</td><td>Nuwacell</td><td>RC02003</td><td>Cell lines</td></tr><tr><td>ncMission</td><td>Shownin</td><td>RP02010</td><td></td></tr><tr><td>Pen Strep</td><td>Gibco</td><td>15140-122</td><td></td></tr><tr><td>pCT-Mito-GFP</td><td>SBI</td><td>CYTO102-PA-1</td><td>Plasmid; From&amp;nbsp; https://www.systembio.com/mitochondria-cyto-tracer-pct-mito-gfp-cmv</td></tr><tr><td>Puromycin</td><td>MCE</td><td>HY-B1743A</td><td></td></tr><tr><td>Pipette</td><td>Axygen</td><td>TF-1000-R-S</td><td></td></tr><tr><td>Phalloidin</td><td>Invitrogen</td><td>A22287</td><td></td></tr><tr><td>Triton X-100</td><td>Solarbio</td><td>T8200</td><td></td></tr><tr><td>Transwell plate</td><td>Corning</td><td>3470</td><td></td></tr></tbody></table>

mitochondrial transfer via tunneling nanotubes between mesenchymal stem cells and retinal pigment epithelium in vitro

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.

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...

Author Spotlight: Bidirectional Mitochondrial Transfer between MSCs and Retinal Pigment Epithelium Cells &#8212; Pathways and In Vivo Challenges

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 Via Tunneling Nanotubes Between Mesenchymal Stem Cells and Retinal Pigment Epithelium In Vitro

Mitochondrial transfer is a normal physiological phenomenon that occurs widely among various types of cells. In the study to date, the most important ...

mitochondrial-transfer-via-tunneling-nanotubes-between-mesenchymal

Research

JoVE Journal

Biology

720 Views.  Capital Medical University. Onze studie richtte zich op bidirectionele mitochondriale overdracht tussen MSC en de retinale pigmentepitheelcellen. We onderzoeken de overdracht van mitochondriën tussen cellen en bepalen de verhouding van mitochondriale overdracht in twee verschillende richtingen. Het is moeilijk om TNT-gemedieerde mitochondriale overdracht in vivo te detecteren vanwege de dichte seniorenopstelling en uitdagingen om mitochondriën in te voeren en te volgen.Het is o...

Video: Auteur in de schijnwerpers: bidirectionele mitochondriale overdracht tussen MSC's en retinale pigmentepitheelcellen - routes en in vivo uitdagingen

In Vivo Tracking of Human Adipose-derived Mesenchymal Stem Cells in a Rat Knee Osteoarthritis Model with Fluorescent Lipophilic Membrane Dye

In order to support the clinical application of human adipose-derived mesenchymal stem cell (haMSC) therapy for knee osteoarthritis (KOA), we examined the efficacy of cell persistence and biodistribution of haMSCs in animal models. We demonstrated a method to label the cell membrane of haMSCs with lipophilic fluorescent dye. Subsequently, intra-articular injection of the labeled cells in rats with surgically induced KOA was monitored dynamically by an in vivo imaging system. We employed the lipophilic carbocyanines DiD (DilC18 (5)), a far-red fluorescent Dil (dialkylcarbocyanines) analog, which utilized a red laser to avoid excitation of the natural green autofluorescence from surrounding tissues. Furthermore, the red-shifted emission spectra of DiD allowed deep-tissue imaging in live animals and the labeling procedure caused no cytotoxic effects or functional damage to haMSCs. This approach has been shown to be an efficient tracking method for haMSCs in a rat KOA model. The application of this method could also be used to determine the optimal administration route and dosage of MSCs from other sources in pre-clinical studies.

In Vivo Tracking of Human Adipose-derived Mesenchymal Stem Cells in a Rat Knee Osteoarthritis Model with Fluorescent Lipophilic Membrane Dye

This protocol describes an efficient way to monitor the cell persistence and biodistribution of human adipose-derived mesenchymal stem cells (haMSCs) by far-red fluorescence labeling in a rat knee osteoarthritis (KOA) model via intra-articular (IA) injection.

In order to support the clinical application of human adipose-derived mesenchymal stem cell (haMSC) therapy for knee osteoarthritis (KOA), we examined the ...

Developmental Biology

Subretinal Transplantation of MACS Purified Photoreceptor Precursor Cells into the Adult Mouse Retina

Vision impairment and blindness due to the loss of the light-sensing cells of the retina, i.e. photoreceptors, represents the main reason for disability in industrialized countries. Replacement of degenerated photoreceptors by cell transplantation represents a possible treatment option in future clinical applications. Indeed, recent preclinical studies demonstrated that immature photoreceptors, isolated from the neonatal mouse retina at postnatal day 4, have the potential to integrate into the adult mouse retina following subretinal transplantation. Donor cells generated a mature photoreceptor morphology including inner and outer segments, a round cell body located at the outer nuclear layer, and synaptic terminals in close proximity to endogenous bipolar cells. Indeed, recent reports demonstrated that donor photoreceptors functionally integrate into the neural circuitry of host mice. For a future clinical application of such cell replacement approach, purified suspensions of the cells of choice have to be generated and placed at the correct position for proper integration into the eye. For the enrichment of photoreceptor precursors, sorting should be based on specific cell surface antigens to avoid genetic reporter modification of donor cells. Here we show magnetic-associated cell sorting (MACS) - enrichment of transplantable rod photoreceptor precursors isolated from the neonatal retina of photoreceptor-specific reporter mice based on the cell surface marker CD73. Incubation with anti-CD73 antibodies followed by micro-bead conjugated secondary antibodies allowed the enrichment of rod photoreceptor precursors by MACS to approximately 90%. In comparison to flow cytometry, MACS has the advantage that it can be easier applied to GMP standards and that high amounts of cells can be sorted in relative short time periods. Injection of enriched cell suspensions into the subretinal space of adult wild-type mice resulted in a 3-fold higher integration rate compared to unsorted cell suspensions.

Cell transplantation represents a strategy for the treatment of retinal degeneration characterized by photoreceptor loss. Here we describe a method for enrichment of transplantable photoreceptors and their subretinal grafting into adult mice.

Vision impairment and blindness due to the loss of the light-sensing cells of the retina, i.e. photoreceptors, represents the main reason for disability ...

Medicine

Engineering Transplantation-suitable Retinal Pigment Epithelium Tissue Derived from Human Embryonic Stem Cells

Several pathological conditions of the eye affect the functionality and/or the survival of the retinal pigment epithelium (RPE). These include some forms of retinitis pigmentosa (RP) and age-related macular degeneration (AMD). Cell therapy is one of the most promising therapeutic strategies proposed to cure these diseases, with already encouraging preliminary results in humans. However, the method of preparation of the graft has a significant impact on its functional outcomes in vivo. Indeed, RPE cells grafted as a cell suspension are less functional than the same cells transplanted as a retinal tissue. Herein, we describe a simple and reproducible method to engineer RPE tissue and its preparation for an in vivo implantation. RPE cells derived from human pluripotent stem cells are seeded on a biological support, the human amniotic membrane (hAM). Compared to artificial scaffolds, this support has the advantage of having a basement membrane that is close to the Bruch&#39;s membrane where endogenous RPE cells are attached. However, its manipulation is not easy, and we developed several strategies for its proper culturing and preparation for grafting in vivo.

We describe a method to engineer a retinal tissue composed of retinal pigment epithelial cells derived from human pluripotent stem cells cultured on top of human amniotic membranes and its preparation for grafting in animal models.

Several pathological conditions of the eye affect the functionality and/or the survival of the retinal pigment epithelium (RPE). These include some forms ...

Performing Subretinal Injections in Rodents to Deliver Retinal Pigment Epithelium Cells in Suspension

The conversion of light into electrical impulses occurs in the outer retina and is accomplished largely by rod and cone photoreceptors and retinal pigment epithelium (RPE) cells. RPE provide critical support for photoreceptors and death or dysfunction of RPE cells is characteristic of age-related macular degeneration (AMD), the leading cause of permanent vision loss in people age 55 and older. While no cure for AMD has been identified, implantation of healthy RPE in diseased eyes may prove to be an effective treatment, and large numbers of RPE cells can be readily generated from pluripotent stem cells. Several interesting questions regarding the safety and efficacy of RPE cell delivery can still be examined in animal models, and well-accepted protocols used to inject RPE have been developed. The technique described here has been used by multiple groups in various studies and involves first creating a hole in the eye with a sharp needle. Then a syringe with a blunt needle loaded with cells is inserted through the hole and passed through the vitreous until it gently touches the RPE. Using this injection method, which is relatively simple and requires minimal equipment, we achieve consistent and efficient integration of stem cell-derived RPE cells in between the host RPE that prevents significant amount of photoreceptor degeneration in animal models. While not part of the actual protocol, we also describe how to determine the extent of the trauma induced by the injection, and how to verify that the cells were injected into the subretinal space using in vivo imaging modalities. Finally, the use of this protocol is not limited to RPE cells; it may be used to inject any compound or cell into the subretinal space.

Here we present a community accepted protocol in multimedia format for subretinally injecting a bolus of RPE cells in rats and mice. This approach can be used for determining rescue potentials, safety profiles, and survival capacities of grafted RPE cells upon implantation in animal models of retinal degeneration.

The conversion of light into electrical impulses occurs in the outer retina and is accomplished largely by rod and cone photoreceptors and retinal pigment ...

Mitochondrial Transfer from Mouse Adipose-Derived Mesenchymal Stem Cells into Aged Mouse Oocytes

Due to the decline in the quantity and quality of oocytes related to age, the fertility of women over 35 years of age has declined sharply. The molecular mechanisms that maintain oocyte quality remain unclear, thus it is difficult to increase the birth rate of women over 35 years old at present. Oocytes contain more mitochondria than any type of cell in the body, and any mitochondrial dysfunction can lead to reduced oocyte quality. In the 1990s, oocyte cytoplasmic transfer resulted in great success in human reproduction but was accompanied by ethical controversies. Autologous mitochondrial transplantation is expected to be a useful technique to increase the quality of oocytes that have decreased due to age. In the present study, we used adipose-derived stem cells from aged mice as a mitochondria donor to increase the quality of oocytes of aged mice. Further development of autologous mitochondrial transfer technology will provide a new and effective treatment for infertility in aged women.

Here, we describe the isolation of mitochondria from mouse adipose-derived mesenchymal stem cells, and then transfer the mitochondria into aged mouse oocytes to improve the quality of the oocytes.

Due to the decline in the quantity and quality of oocytes related to age, the fertility of women over 35 years of age has declined sharply. The molecular ...

Flow Cytometric Analysis of Multiple Mitochondrial Parameters in Human Induced Pluripotent Stem Cells and Their Neural and Glial Derivatives

Mitochondria are important in the pathophysiology of many neurodegenerative diseases. Changes in mitochondrial volume, mitochondrial membrane potential (MMP), mitochondrial production of reactive oxygen species (ROS), and mitochondrial DNA (mtDNA) copy number are often features of these processes. This report details a novel flow cytometry-based approach to measure multiple mitochondrial parameters in different cell types, including human induced pluripotent stem cells (iPSCs) and iPSC-derived neural and glial cells. This flow-based strategy uses live cells to measure mitochondrial volume, MMP, and ROS levels, as well as fixed cells to estimate components of the mitochondrial respiratory chain (MRC) and mtDNA-associated proteins such as mitochondrial transcription factor A (TFAM). 
By co-staining with fluorescent reporters, including MitoTracker Green (MTG), tetramethylrhodamine ethyl ester (TMRE), and MitoSox Red, changes in mitochondrial volume, MMP, and mitochondrial ROS can be quantified and related to mitochondrial content. Double staining with antibodies against MRC complex subunits and translocase of outer mitochondrial membrane 20 (TOMM20) permits the assessment of MRC subunit expression. As the amount of TFAM is proportional to mtDNA copy number, the measurement of TFAM per TOMM20 gives an indirect measurement of mtDNA per mitochondrial volume. The entire protocol can be carried out within 2-3 h. Importantly, these protocols allow the measurement of mitochondrial parameters, both at the total level and the specific level per mitochondrial volume, using flow cytometry.

This study reports a novel approach to measure multiple mitochondrial functional parameters based on flow cytometry and double staining with two fluorescent reporters or antibodies to detect changes in mitochondrial volume, mitochondrial membrane potential, reactive oxygen species level, mitochondrial respiratory chain composition, and mitochondrial DNA.

Mitochondria are important in the pathophysiology of many neurodegenerative diseases. Changes in mitochondrial volume, mitochondrial membrane potential ...

Neuroscience

Transpupillary-Guided Trans-Scleral Transplantation of Subretinal Grafts in a Retinal Degeneration Mouse Model

Transplantation of photoreceptor cells and retinal pigment&#160;epithelial (RPE) cells provide a potential therapy for retinal degeneration diseases. Subretinal transplantation of therapeutic donor cells into mouse recipients is challenging due to the limited surgical space allowed by the small volume of the mouse eye. We developed a trans-scleral surgical transplantation platform with direct transpupillary vision guidance to facilitate the subretinal delivery of exogenous cells in mouse recipients. The platform was tested using retinal cell suspensions and three-dimensional retinal sheets collected from rod-rich Rho::EGFP mice and cone-rich OPN1LW-EGFP;NRL-/- mice, respectively. Live/dead assay showed low cell mortality for both forms of donor cells. Retinal grafts were successfully delivered into the subretinal space of a mouse model of retinal degeneration, Rd1/NS, with minimum surgical complications as detected by multimodal confocal scanning laser ophthalmoscope (cSLO) imaging. Two months post-transplantation, histological staining demonstrated evidence of advanced maturation of the retinal grafts into &#39;adult&#39; rods and cones (by robust Rho::EGFP, S-opsin, and OPN1LW:EGFP expression, respectively) in the subretinal space. Here, we provide a surgical platform that can enable highly accurate subretinal delivery with a low rate of complications in mouse recipients. This technique offers precision and relative ease of skill acquisition. Furthermore, the technique could be used not only for studies of subretinal cell transplantation but also for other intraocular therapeutic studies including gene therapies.

This protocol presents a transpupillary vision-guided trans-scleral approach to safely and precisely deliver subretinal cellular grafts, with a low rate of surgical complications, in mouse recipients with or without retinal degeneration.

Transplantation of photoreceptor cells and retinal pigment&#160;epithelial (RPE) cells provide a potential therapy for retinal degeneration diseases. ...

MitoCeption: Transferring Isolated Human MSC Mitochondria to Glioblastoma Stem Cells

Mitochondria play a central role for cell metabolism, energy production and control of apoptosis. Inadequate mitochondrial function has been found responsible for very diverse diseases, ranging from neurological pathologies to cancer. Interestingly, mitochondria have recently been shown to display the capacity to be transferred between cell types, notably from human mesenchymal stem cells (MSC) to cancer cells in coculture conditions, with metabolic and functional consequences for the mitochondria recipient cells, further enhancing the current interest for the biological properties of these organelles.
Evaluating the effects of the transferred MSC mitochondria in the target cells is of primary importance to understand the biological outcome of such cell-cell interactions. The MitoCeption protocol described here allows the transfer of the mitochondria isolated beforehand from the donor cells to the target cells, using MSC mitochondria and glioblastoma stem cells (GSC) as a model system. This protocol has previously been used to transfer mitochondria, isolated from MSCs, to adherent MDA-MB-231 cancer cells. This mitochondria transfer protocol is adapted here for GSCs that present the specific particularity of growing as neurospheres in vitro. The transfer of the isolated mitochondria can be followed by fluorescence-activated cell sorting (FACS) and confocal imaging using mitochondria vital dyes. The use of mitochondria donor and target cells with distinct haplotypes (SNPs) also allows detection of the transferred mitochondria based on the concentration of their circular mitochondrial DNA (mtDNA) in the target cells. Once the protocol has been validated with these criteria, the cells harboring the transferred mitochondria can be further analyzed to determine the effects of the exogenous mitochondria on biological properties such as cell metabolism, plasticity, proliferation and response to therapy.

Here, a protocol (MitoCeption) is presented to transfer mitochondria, isolated from human mesenchymal stem cells (MSC), to glioblastoma stem cells (GSC), with the goal of studying their biological effects on GSC metabolism and functions. A similar protocol can be adapted to transfer mitochondria between other cell types.

Mitochondria play a central role for cell metabolism, energy production and control of apoptosis. Inadequate mitochondrial function has been found ...

Revitalizing Sight: Stem Cell Therapy for Retinal Degeneration

Sub-Retinal Delivery of Human Embryonic Stem Cell Derived Photoreceptor Progenitors in rd10 Mice

Regeneration of photoreceptor cells using human pluripotent stem cells is a promising therapy for the treatment of both hereditary and aging retinal diseases at advanced stages. We have shown human recombinant retina-specific laminin isoform matrix is able to support the differentiation of human embryonic stem cells (hESCs) to photoreceptor progenitors. In addition, sub-retinal injection of these cells has also shown partial restoration in the rd10 rodent and rabbit models. Sub-retinal injection is known to be an established method that has been used to deliver pharmaceutical compounds to the photoreceptor cells and retinal pigmented epithelial (RPE) layer of the eye due to its proximity to the target space. It has also been used to deliver adeno-associated viral vectors into the sub-retinal space to treat retinal diseases. The sub-retinal delivery of pharmaceutical compounds and cells in the murine model is challenging due to the constraint in the size of the murine eyeball. This protocol describes the detailed procedure for the preparation of hESC-derived photoreceptor progenitor cells for injection and the sub-retinal delivery technique of these cells in genetic retinitis pigmentosa mutant, rd10 mice. This approach allows cell therapy to the targeted area, in particular the outer nuclear layer of the retina, where diseases leading to photoreceptor degeneration occur.

Author Spotlight: Advancing Vision Restoration - Stem Cell-Based Therapy for Retinal Diseases 

We describe a detailed protocol for the preparation of post-cryopreserved hESC-derived photoreceptor progenitor cells and the sub-retinal delivery of these cells in rd10 mice.

Regeneration of photoreceptor cells using human pluripotent stem cells is a promising therapy for the treatment of both hereditary and aging retinal ...

Centrifugation-Assisted Mitochondrial Transfer to Glioblastoma Stem Cells

Source: Mombo, B. N., et al.&#160;<a href="https://app.jove.com/v/55245">MitoCeption: transferring isolated human MSC mitochondria to glioblastoma stem cells.</a> J. Vis. Exp. (2017)
In this video, a method is demonstrated to enable the transfer of fluorescently labeled mitochondria into glioblastoma stem cells (GSCs) through centrifugation. The successful uptake and functional integration of the donor mitochondria are confirmed by detecting the fluoresce using confocal microscopy.

Source: Mombo, B. N., et al.&#160;MitoCeption: transferring isolated human MSC mitochondria to glioblastoma stem cells. J. Vis. Exp. (2017)
In this video, ...

Mitochondrial Transfer Via Tunneling Nanotubes Between Mesenchymal Stem Cells and Retinal Pigment Epithelium In Vitro

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