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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#160; 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 ...

Our study focused on bi-direction mitochondrial transfer between MSC and the retinal pigment epithelial cells. We are investigating the transfer of mitochondria between cells and determining the ratio of mitochondrial transfer in two different directions. It is difficult to detect TNT-mediated mitochondrial transfer in vivo due to the dense senior arrangement and challenges to in and tracking mitochondria.It is also difficult to label the mitochondria of two different cells with different colors. Our previous study observed the phenomena of TNT-mediated mitochondrial transfer by co-capturing MSC in retinal cells, including corneal endothelial cells, photoreceptors, and retinal pigment signal cells in vitro. Functional mitochondrial transfer from MSC can rescue damaged and retinal cells.

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Research

JoVE Journal

Biology

583 Views.  Capital Medical University. Our study focused on bi-direction mitochondrial transfer between MSC and the retinal pigment epithelial cells. We are investigating the transfer of mitochondria between cells and determining the ratio of mitochondrial transfer in two different directions. It is difficult to detect TNT-mediated mitochondrial transfer in vivo due to the dense senior arrangement and challenges to in and tracking mitochondria.It is also difficult to label the mitochondria of two different cells with differ...

Video: Author Spotlight: Bidirectional Mitochondrial Transfer between MSCs and Retinal Pigment Epithelium Cells — Pathways and In Vivo Challenges

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

MicroRNA Expression Profiles of Human iPS Cells, Retinal Pigment Epithelium Derived From iPS, and Fetal Retinal Pigment Epithelium

The objective of this report is to describe the protocols for comparing the microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, retinal pigment epithelium (RPE) derived from human iPS cells (iPS-RPE), and fetal RPE. The protocols include collection of RNA for analysis by microarray, and the analysis of microarray data to identify miRNAs that are differentially expressed among three cell types. The methods for culture of iPS cells and fetal RPE are explained. The protocol used for differentiation of RPE from human iPS is also described. The RNA extraction technique we describe was selected to allow maximal recovery of very small RNA for use in a miRNA microarray. Finally, cellular pathway and network analysis of microarray data is explained. These techniques will facilitate the comparison of the miRNA profiles of three different cell types.

The microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, retinal pigment epithelium (RPE) derived from human induced-pluripotent stem (iPS) cells (iPS-RPE), and fetal RPE, were compared.

The objective of this report is to describe the protocols for comparing the microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, ...

Primary Cell Cultures from the Mouse Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) is a highly polarized multi-functional epithelium that is located between the neural retina and the choroid of the eye. It is a single sheet of pigmented cells that are hexagonally packed and connected by tight junctions. The main functions of the RPE include absorption of light, phagocytosis of the shed photoreceptor outer segments, spatial buffering of ions, transport of nutrients, ions and water as well as active involvement in the visual cycle. With such important and diverse functions, it is critically important to study the biology of RPE cells. A number of RPE cell lines have been established; however, passaged and immortalized cells are known to quickly lose some of the morphological and physiological characteristics of natural RPE cells. Thus, primary cells are more suitable for studying different aspects of RPE cell biology and function. Mouse primary RPE cell culture is very useful to researchers since mouse models are widely used in biological studies, however collecting RPE cells from mouse is also very challenging due to their small size. Here, we present a protocol for establishing primary mouse RPE cell cultures which includes enucleation and dissection of the eyes and isolation of the RPE sheets to yield the cells for culturing. This method enables efficient cell recovery. The RPE cells obtained from two mice&#160;can reach confluency on one 12 mm polyester membrane insert pre-loaded in culture plate after one week of culture and display some of the original properties of bona fide RPE cells such as hexagonal shape and pigmentation after two weeks of culture.

The retinal pigment epithelium (RPE) is a multi-functional epithelium of the eye. Here we present a protocol to establish primary cell cultures derived from the murine RPE.

The retinal pigment epithelium (RPE) is a highly polarized multi-functional epithelium that is located between the neural retina and the choroid of the ...

Detection and Quantification of Tunneling Nanotubes Using 3D Volume View Images

Recent discoveries have revealed that cells perform direct, long-range, intercellular transfer via nano-scale, actin-membrane conduits, namely &#34;tunneling nanotubes&#34; (TNTs). TNTs are defined as open-ended, lipid bilayer-encircled membrane extensions that mediate continuity between neighboring cells of diameters ranging between 50 nm and 1 &#181;m. TNTs were demonstrated initially in neuronal cells, but successive studies have revealed the existence of TNTs in several cell types and diseases, such as neurodegenerative diseases, viral infections, and cancer. Several studies have referred to close-ended, electrically coupled membrane nanostructures between neighboring cells as TNTs or TNT-like structures.
The elucidation of ultrastructure in terms of membrane continuity at the endpoint is technically challenging. In addition, studies on cell-cell communication are challenging in terms of the characterization of TNTs using conventional methods due to the lack of specific markers. TNTs are primarily defined as F-actin-based, open-ended membrane protrusions. However, one major limitation is that F-actin is present in all types of protrusions, making it challenging to differentiate TNTs from other protrusions. One of the notable characteristics of F-actin-based TNTs is that these structures hover between two cells without touching the substratum. Therefore, distinct F-actin-stained TNTs can conveniently be distinguished from other protrusions such as filopodia and neurites based on their hovering between cells.
We have recently shown that the internalization of oligomeric amyloid-&#946;1-42 (oA&#946;) via actin-dependent endocytosis stimulates activated p21-activated kinase-1 (PAK1), which mediates the formation of F-actin-containing TNTs coexpressed with phospho-PAK1 between SH-SY5Y neuronal cells. This protocol outlines a 3D volume analysis method to identify and characterize TNTs from the captured z-stack images of F-actin- and phospho-PAK1-immunostained membrane protrusions in oA&#946;-treated neuronal cells. Further, TNTs are distinguished from developing neurites and neuronal outgrowths based on F-actin- and &#946;-III tubulin-immunostained membrane conduits.

Tunneling nanotubes (TNTs) are primarily open-ended F-actin membrane nanotubes that connect neighboring cells, facilitating intercellular communication. The notable characteristic that distinguishes TNTs from other cell protrusions is the hovering nature of the nanotubes between cells. Here, we characterize TNTs by constructing a 3D volume view of confocal z-stack images.

Recent discoveries have revealed that cells perform direct, long-range, intercellular transfer via nano-scale, actin-membrane conduits, namely ...

Primary Culture of Porcine Retinal Pigment Epithelial Cells

The retinal pigment epithelium (RPE) is a monolayer of polarized pigmented epithelial cells, located between the choroid and neuroretina in the retina. Multiple functions, including phagocytosis, nutrient/metabolite transportation, vitamin A metabolism, etc., are conducted by the RPE on a daily basis. RPE cells are terminally differentiated epithelial cells with little or no regenerative capacity. Loss of RPE cells results in multiple eye diseases leading to visual impairment, such as age-related macular degeneration. Therefore, the establishment of an in vitro culture model of primary RPE cells, which more closely resembles the RPE in vivo than cell lines, is critical for the characteristic and mechanistic studies of RPE cells. Considering the fact that the source of human eyeballs is limited, we create a protocol to culture primary porcine RPE cells. By using this protocol, RPE cells can be easily dissociated from adult porcine eyeballs. Subsequently, these dissociated cells attach to culture dishes/inserts, proliferate to form a confluent monolayer, and quickly re-establish key features of epithelial tissue in vivo within 2 wks. By qRT-PCR, it is demonstrated that primary porcine RPE cells express multiple signature genes at comparable levels with native RPE tissue, while the expressions of most of these genes are lost/highly reduced in human RPE-like cells, ARPE-19. Moreover, the immunofluorescence staining shows the distribution of tight junction, tissue polarity, and cytoskeleton proteins, as well as the presence of RPE65, an isomerase critical for vitamin A metabolism, in cultured primary cells. Altogether, we have developed an easy-to-follow approach to culture primary porcine RPE cells with high purity and native RPE features, which could serve as a good model to understand RPE physiology, study cell toxicities, and facilitate drug screenings.

Here, an easy-to-follow method to culture primary porcine retinal pigment epithelial cells in vitro is presented.

The retinal pigment epithelium (RPE) is a monolayer of polarized pigmented epithelial cells, located between the choroid and neuroretina in the retina. ...

Isolation of Primary Mouse Retinal Pigmented Epithelium Cells

The retinal pigmented epithelium (RPE) layer lies immediately behind the photoreceptors and harbors a complex metabolic system that plays several critical roles in maintaining the photoreceptors' function. Thus, the RPE structure and function are essential to sustain normal vision. This manuscript presents an established protocol for primary mouse RPE cell isolation. RPE isolation is a great tool to investigate the molecular mechanisms underlying RPE pathology in the different mouse models of ocular disorders. Furthermore, RPE isolation can help in comparing primary mouse RPE cells isolated from wild-type and genetically modified mice, as well as testing drugs that can accelerate the development of therapy for visual disorders. The manuscript presents a step-by-step RPE isolation protocol; the entire procedure, from enucleation to seeding, takes approximately 4 hours. The media shouldn't be changed for 5-7 days after seeding, to allow the growth of the isolated cells without disturbance. This process is followed by the characterization of morphology, pigmentation, and specific markers in the cells via immunofluorescence. Cells can be passaged a maximum of three or four times.

This manuscript describes a simplified protocol for the isolation of retinal pigmented epithelium (RPE) cells from mouse eyes in a stepwise manner. The protocol includes the enucleation and dissection of mouse eyes, followed by the isolation, seeding, and culturing of RPE cells.

The retinal pigmented epithelium (RPE) layer lies immediately behind the photoreceptors and harbors a complex metabolic system that plays several critical ...

Efficient and Consistent Generation of Retinal Pigment Epithelium/Choroid Flatmounts from Human Eyes for Histological Analysis

The retinal pigment epithelium (RPE) and retina are functionally and structurally connected tissues that work together to regulate light perception and vision. Proteins on the RPE apical surface are tightly associated with proteins on the photoreceptor outer segment surface, making it difficult to consistently separate the RPE from the photoreceptors/retina. We developed a method to efficiently separate the retina from the RPE of human eyes to generate complete RPE/choroid and retina flatmounts for separate cellular analysis of the photoreceptors and RPE cells. An intravitreal injection of a high-osmolarity solution of D-mannitol, a sugar not transported by the RPE, induced the separation of the RPE and retina across the entire posterior chamber without causing damage to the RPE cell junctions. No RPE patches were observed attached to the retina. Phalloidin labeling of actin showed RPE shape preservation and allowed morphometric analysis of the entire epithelium. An artificial intelligence (AI)-based software was developed to accurately recognize and segment the RPE cell borders and quantify 30 different shape metrics. This dissection method is highly reproducible and can be easily extended to other animal models.

We describe a method to efficiently separate retinal pigment epithelium (RPE) from the retina in human eyes and generate whole RPE/choroid flatmounts for histological and morphometric analyses of the RPE.

The retinal pigment epithelium (RPE) and retina are functionally and structurally connected tissues that work together to regulate light perception and ...

Modeling and Analyzing Retinal Degeneration

LipidUNet-Machine Learning-Based Method of Characterization and Quantification of Lipid Deposits Using iPSC-Derived Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) is a monolayer of hexagonal cells located at the back of the eye. It provides nourishment and support to photoreceptors and choroidal capillaries, performs phagocytosis of photoreceptor outer segments (POS), and secretes cytokines in a polarized manner for maintaining the homeostasis of the outer retina. Dysfunctional RPE, caused by mutations, aging, and environmental factors, results in the degeneration of other retinal layers and causes vision loss. A hallmark phenotypic feature of degenerating RPE is intra and sub-cellular lipid-rich deposits. These deposits are a common phenotype across different retinal degenerative diseases. To reproduce the lipid deposit phenotype of monogenic retinal degenerations in vitro, induced pluripotent stem cell-derived RPE (iRPE) was generated from patients&#39; fibroblasts. Cell lines generated from patients with Stargardt and Late-onset retinal degeneration (L-ORD) disease were fed with POS for 7 days to replicate RPE physiological function, which caused POS phagocytosis-induced pathology in these diseases. To generate a model for age-related macular degeneration (AMD), a polygenic disease associated with alternate complement activation, iRPE was challenged with alternate complement anaphylatoxins. The intra and sub-cellular lipid deposits were characterized using Nile Red, boron-dipyrromethene (BODIPY), and apolipoprotein E (APOE). To quantify the density of lipid deposits, a machine learning-based software, LipidUNet, was developed. The software was trained on maximum-intensity projection images of iRPE on culture surfaces. In the future, it will be trained to analyze three-dimensional (3D) images and quantify the volume of lipid droplets. The LipidUNet software will be a valuable resource for discovering drugs that decrease lipid accumulation in disease models.

Author Spotlight: Unraveling the Pathogenesis of Age-Related Macular Degeneration and Discovering Potential Therapies 

Degenerative eye diseases that affect the retinal pigment epithelium layer of the eye have monogenic and polygenic origins. Several disease models and a software application, LipidUNet, have been developed to study mechanisms of disease, as well as potential therapeutic interventions.

The retinal pigment epithelium (RPE) is a monolayer of hexagonal cells located at the back of the eye. It provides nourishment and support to ...

A Cost-Effective Method for Isolating Primary RPE Cells from Porcine Eyes

Isolation of Primary Porcine Retinal Pigment Epithelial Cells for In Vitro Modeling

The retinal pigment epithelium (RPE) is a crucial monolayer in the outer retina responsible for supporting photoreceptors. RPE degeneration commonly occurs in diseases marked by progressive vision loss, such as age-related macular degeneration (AMD). Research on AMD often relies on human donor eyes or induced pluripotent stem cells (iPSCs) to represent the RPE. However, these RPE sources require extended differentiation periods and substantial expertise for culturing. Additionally, some research institutions, particularly those in rural areas, lack easy access to donor eyes. While a commercially available immortalized RPE cell line (ARPE-19) exists, it lacks essential in vivo RPE features and is not widely accepted in many ophthalmology research publications. There is a pressing need to obtain representative primary RPE cells from a more readily available and cost-effective source. This protocol elucidates the isolation and subculture of primary RPE cells obtained post-mortem from porcine eyes, which can be sourced locally from commercial or academic suppliers. This protocol necessitates common materials typically found in tissue culture labs. The result is a primary, differentiated, and cost-effective alternative to iPSCs, human donor eyes, and ARPE-19 cells.

Author Spotlight: Modeling Retinal Pathologies with Enhanced RPE Cell-Based Disease Models

This protocol outlines the procedure for obtaining and culturing primary retinal pigment epithelial (RPE) cells from locally sourced porcine eyes. These cells serve as a high-quality alternative to stem cells and are suitable for in vitro retinal research.

The retinal pigment epithelium (RPE) is a crucial monolayer in the outer retina responsible for supporting photoreceptors. RPE degeneration commonly ...