This work was supported by grants from the National Health Research Institutes (05A1-CSPP16-014, H.J.L.) and from the Ministry of Science and Technology (MOST 103-2320-B-400-015-MY3, H.J.L).

All research involving animals complied with protocols approved by the Institutional Committee on Animal Care.
1. Extracellular Vesicle/exosome Isolation and Validation
<ol>
	<li>Culture mammary epithelial basal cells, NAMECs4, with fresh, serum-free medium made of 500 mL of MCDB 170, pH 7.4 + 500 mL of DMEM/F12 with sodium bicarbonate (0.2438%); EGF (5 ng/mL); hydrocoritisone (0.5 &#181;g/mL); insulin (5 &#181;g/mL); bovine pituitary extract (BPE; 35 &#181;g/mL); and GW627368X (1 &#181;g/mL) in 15 cm dishes.</li>
	<li>After counting the cells with a hemocytometer, seed 1.2 x 106 cells in 12 mL of medium per 15-cm dish on day 0 for 4 days4.</li>
	<li>After 4 days in culture, centrifuge the culture medium at 300 x g for 5 min using a table-top centrifuge. Transfer the supernatant to a conical tube (Figure 1).</li>
	<li>Centrifuge the supernatant at 2,000 x g for 20 min in a table-top centrifuge. Transfer the supernatant to an ultracentrifuge tube and leave the dead cells and cell debris (Figure 1).</li>
	<li>Centrifuge the supernatant at 10,000 x g for 30 min at 4 &#176;C. Transfer the supernatant to a new ultracentrifuge tube (Figure 1).</li>
	<li>Centrifuge the supernatant at 110,000 x g for 60 min at 4 &#176;C. Remove the supernatant and resuspend the EV/exosome pellet in PBS (Figure 1).</li>
	<li>Centrifuge the supernatant at 110,000 x g for 60 min at 4 &#176;C. Remove the supernatant. Resuspend the EV/exosome pellet in PBS (Figure 1). Resuspend the pellet isolated from 240-480 mL of NAMEC-conditioned medium in 100 &#181;L.</li>
	<li>Measure the protein concentration of the EV suspension with the BCA protein assay. Ensure that the concentration is around 20-40 &#181;g/100 &#181;L. Store at -20 &#176;C for further analysis.</li>
	<li>Measure the concentration and size of the EVs/exosomes by nanoparticle tracking analysis (NTA), as described previously by Gardiner et al.11. Dilute the EVs/exosomes (20 &#181;g/100 &#181;L) with PBS to 10,000-fold for NTA analysis. 
	NOTE: The result of NTA analysis reflects the number and size of the vesicles analyzed.</li>
	<li>Image the EVs/exosomes with transmissionelectron microscopy (TEM), as described previously by Lin et al4.</li>
</ol>
2. Exosome Purification Using a Density Gradient
<ol>
	<li>Resuspend the 110,000 g pellet from step 1.7 in 40% (w/v) iodixanol in PBS (2 mL). Overlay the mixture in sequence with aliquots of 30%, 20%, 10%, and 5% (w/v) iodixanol in PBS (2 mL each) to form a density gradient in an ultracentrifuge tube.</li>
	<li>Centrifuge the mixture at 200,000 x g for 8 h at 4 &#176;C.</li>
	<li>Collect each gradient fraction (10 fractions; 1 mL/fraction) with a pipette from the top of the tube.</li>
	<li>Analyze the presence of exosome markers (e.g., CD81, CD9, CD63, and Tsg101) in each fraction by SDS-PAGE12 and Western blot. Load 50-&#181;L suspensions of each fraction onto a 10% gel containing 0.1% (w/v) SDS and separate the proteins in fractions with gel electrophoresis.</li>
	<li>Transfer the proteins from a gel to a PVDF membrane and incubate the membrane with antibodies against the exosome markers (e.g., CD81, CD9, CD63, and Tsg101) and housekeeping protein GAPDH overnight (Table of Materials) at 4 &#176;C. 
	NOTE: The result identifies the fraction containing exosomes.</li>
</ol>
3. Extracellular Vesicle/Exosome Labeling
<ol>
	<li>Suspend the EVs/exosomes, obtained in step 1.7, in 10 &#181;M carboxyfluorescein succinimidyl diacetate ester (CFSE) at 20 &#181;g of exosomal protein/100 &#181;L. Prepare a parallel sample containing only CFSE and PBS, processed in the same manner, as a negative control for the later EV/exosome uptake assays. Leave the mixtures at 37 &#176;C for 30 min.</li>
	<li>Suspend the EVs/exosomes in 50x volume of PBS and centrifuge the suspension at 110,000 x g for 60 min at 4 &#176;C. Remove the supernatant and resuspend the EV/exosome pellet in PBS. Repeat step 3.2 once.</li>
	<li>Suspend the EVs/exosomes in PBS at a concentration of 20 &#181;g of exosomal protein/100 &#181;L and then filter the EVs/exosomes through 0.22-&#181;m membranes before adding the EVs/exosomes to the cells.</li>
</ol>
4. Extracellular Vesicle/Exosome Uptake Assay
<ol>
	<li>To make culture medium, mix 500 mL of MCDB 170, pH 7.4 + 500 mL of DMEM/F12 with sodium bicarbonate (0.2438%); EGF (5 ng/mL); hydrocoritisone (0.5 &#181;g/mL); insulin (5 &#181;g/mL); and BPE (35 &#181;g/mL)4. Plate the human mammary epithelial HMLE cells in 6-well dishes (1 x 106 cells/well) one day before EV/exosome treatment. Add 2 &#181;g/mL CFSE fluorescence-labeled EVs/exosome, obtained in step 3.3, to the culture medium of the HMLE cells for 2-6 h. Treat the HMLE cells of the negative control group with the parallel preparation, described in step 3.1.</li>
	<li>After the 2- to 6-h incubation, wash the cells twice with 4 mL of PBS at room temperature.</li>
	<li>Detach the cells with 0.25% trypsin for 10 min and re-suspend the cells in PBS containing 0.2% FBS. Measure the EV/exosome uptake from the fluorescence intensity in the cells using a fluorescence cell analyzer4. Image the cells treated with EVs/exosomes or the negative control using confocal microscopy. 
	NOTE: The green fluorescence in the cells is caused by the EV/exosome uptake. See the legend of Figure 6 for the microscope settings.</li>
</ol>
5. Isolation of Primary Mouse Mammary Epithelial Cells
<ol>
	<li>Prepare gelatin-coated dishes by adding 12 mL of 0.1% gelatin solution to 10 cm dishes. Place the plates in a 37 &#176;C incubator for 30 min. Remove the gelatin solution and leave the lids off the dishes in a laminar flow hood for 4 h until the gelatin coating has dried.</li>
	<li>Dissect the number 2, 3, 4, and 5 mammary glands (Figure 2) from 12-week-old virgin female C57BL/6 mice using scissors and cut the glands into small pieces (2 mm2) using a razor.</li>
	<li>Further dissociate the slurry of mammary glands (from 10 mice) for 60 min at 37 &#176;C, 120 rpm agitation with 50 mL of DMEM/F12 containing 0.2% collagenase type IV, 0.2% trypsin, 5% FBS, 5 &#181;g/mL gentamycin, and 1x pen-strep.</li>
	<li>Pellet down the epithelial organoids from the mixture by centrifugation at 350 x g for 10 min.</li>
	<li>Suspend the epithelial organoids in 4 mL of DMEM/F12 with 0.1 mg/mL DNase I for 5 min at room temperature. Add 6 mL of DMEM/F12 to a final volume of 10 mL.</li>
	<li>Centrifuge the suspension at 400 x g for 10 min at room temperature and discard the supernatant.</li>
	<li>Resuspend the pellet in 10 mL of DMEM/F12. Centrifuge the suspension but hit the brake when the speed reaches 400 x g. Discard the supernatant. 
	NOTE: By hitting the brake, epithelial organoids are quickly pelleted down, while fibroblasts, as single cells, still stay in the supernatant.</li>
	<li>Repeat step 5.6 and 5.7 5-7 times. Add a drop of the suspension to a hemocytometer and then check the clearance of fibroblasts from the organoid mixture under microscopy after each round of centrifugation (Figure 3).</li>
	<li>Plate the organoids in the gelatin-coated dish, obtained in step 5.1, for 48 h with DMEM/F12 containing 1x ITS, 5% FBS, 50 &#181;g/mL gentamycin, 10 ng/mL EGF, and 1x pen-strep.</li>
	<li>After 48 h, remove the floating cells in the culture by replacing the medium with fresh culture medium without FBS (DMEM/F12 containing 1x ITS, 50 &#181;g/mL gentamycin, 10 ng/mL EGF, and 1x pen-strep).</li>
	<li>Confirm that a monolayer of mammary epithelial cells migrates and grows out from the attached epithelial organoids in 3 days.</li>
</ol>
6. Separation of Primary Mouse Basal/Luminal Mammary Epithelial Cells
<ol>
	<li>After the 3-day culture, detach the mouse primary mammary cells with a natural enzyme mixture with proteolytic and collagenolytic enzyme activity for 20 min. Neutralize the enzyme activity with 5% FBS in PBS.</li>
	<li>Centrifuge the suspension at 450 x g for 10 min at room temperature and discard the supernatant. Resuspend the cell pellet in 5 mg/mL dispase for 20 min. Neutralize the enzyme activity with 5% FBS in PBS.</li>
	<li>Centrifuge the suspension at 450 x g for 10 min at room temperature and discard the supernatant.</li>
	<li>Re-suspended the cells in 100 &#181;L PBS with 0.2% FBS at a concentration of 107 cells/mL with the diluted anti-CD49f and anti-EpCAM antibodies on ice for 1 h in the dark.</li>
	<li>Wash the cells with PBS and then incubate the cells with fluorophore-conjugated secondary antibody on ice for 30 min in the dark.</li>
	<li>Wash and re-suspend the cells in PBS with 0.2% FBS.</li>
	<li>Sort the EpCAMhi/CD49flo luminal mammary epithelial cells on a cell sorter4.</li>
</ol>
7. Extracellular Vesicle/Exosome Treatment
<ol>
	<li>Seed the sorted EpCAMhi/CD49flo luminal mammary epithelial cells from step 6.7 on gelatin-coated 6-well dishes (2 x 105 cell/well) and then treat them with PBS or the 2 &#181;g/mL NAMEC-derived EVs/exosomes obtained in step 1.7.</li>
	<li>To ensure the biological efficacy of the EVs/exosomes in long-term culture treatment, replace the cell culture medium with fresh medium containing PBS or 2 &#181;g/mL NAMEC-derived EVs/exosomes every two days. Do not split the mouse primary luminal cells during the 10-day treatment.</li>
</ol>
8. Fat Pad Injection of Mammary Epithelial Cells
<ol>
	<li>Anesthetize a 3-week-old female C57BL/6 mice with isofluorane 2-3% inhalant.</li>
	<li>Place the anesthetized mouse on its back. Remove the fur on the mid-abdomen with a razor/hair cream and clean the surgery site with three alternating cycles of 70% alcohol and povidone-iodine.</li>
	<li>Make a 1.5 cm vertical incision through the skin along the ventral thoracic-inguinal region with scissors and then alternately expose the right and left 4th mammary fat pads.</li>
	<li>Clear each fat pad by removing gland parenchyma with scissors. Locate the lymph node in the fat pad and then remove the whole gland parenchyma below the lymph node. 
	NOTE: Two-thirds of the fat pad should remain in place.</li>
	<li>Detach the cells obtained from step 7.2 with a natural enzyme mixture (see the Table of Materials) with proteolytic and collagenolytic enzyme activity for 10 min. Neutralize the enzyme activity with 5% FBS in PBS.</li>
	<li>Centrifuge the suspension at 450 x g for 10 min at room temperature and discard the supernatant. Count the cells with a hemocytometer and suspend the cells in PBS at a concentration such that 15 &#181;L contains the desired cell dose (104-102 cell/pad).</li>
	<li>Inject 15 &#181;L of mammary epithelial cell suspension into a fat pad using a 100 &#181;L glass syringe attached to a 27G needle.</li>
	<li>Repeat steps 8.4-8.7 for the fat pad on the other side.</li>
	<li>Close the skin with wound clips.</li>
</ol>
9. Mammary Gland Whole Mount
<ol>
	<li>Euthanize the mouse with CO2 plus cervical dislocation at 8 weeks after the cell injection (Step 8.7).</li>
	<li>Make a vertical incision through the skin layer from the thoracic region to the inguinal region using scissors and then expose both the right and left 4th mammary fat pads. Remove the 4th mammary glands (Figure 2).</li>
	<li>Spread the fat pads on glass microscope slides and fix the fat pads with Kahle&#39;s fixative (4% formaldehyde, 30% EtOH, and 2% glacial acetic acid) at room temperature overnight. 
	Caution: Kahle&#39;s fixative is an irritant. Perform this step in a chemical hood.</li>
	<li>Wash the fat pads in 250 mL of 70% EtOH for 15 min and then in 250 mL of dH2O for 5 min. Stain the fat pads with carmine alum (1 g of carmine and 2.5 g of aluminum potassium sulfate in 500 mL of dH2O) at room temperature overnight.</li>
	<li>Wash the fat pads with 250 mL of 70% EtOH for 15 min, 250 mL of 95% EtOH for 15 min, and 250 mL of 100% EtOH for 15 min.</li>
	<li>Clean the fat pads in xylene for days and stop the xylene incubation when the fat pads become transparent. 
	Caution: Xylene is an irritant. Perform this step in a chemical hood.</li>
	<li>Mount the slides with mounting medium and take images (2,400 dpi) of the fat pads using a digital slide scanner.</li>
</ol>

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The authors have nothing to disclose.

Exosomes often carry characteristics of the cells that released them, and the amount of released exosomes can be induced by stimuli4. The culture medium of cells can be collected and subjected to differential ultracentrifugation for EV/exosome collection (Figure 1). There is currently no general agreement on an ideal method to isolate EVs/exosomes. The optimal method used here has been determined by the downstream application14. Ultracentrifuga...

Exosomes can mediate cellular communication by transferring membrane and cytosolic proteins, lipids, and RNAs between cells1. Exosome-mediated communication has been demonstrated to be involved in many physiological and pathological processes (i.e., antigen presentation, development of tolerance2, and tumor progression3). Exosomes often have contents similar to those of the source cells releasing them. Thus, the exosomes can carry specific cell properties from the source cells and transfer these properties to the cells ingesting them4.
Exosomes are 50- to 150-nm double-layer membrane vesicles and present specific markers (e.g., CD9, CD81, CD63, HSP70, Alix, and TSG101). Thus, exosomes must be characterized by various methods for different aspects. Transmission electron microscopy can be used to visualize membrane vesicles such as exosomes4,5. Nanoparticle tracking analysis (NTA) and dynamic light scattering analysis (DLS) are used for measuring the size and number of purified exosomes4. The lipid membrane content of exosomes can be verified by density gradient. Exosomal markers, such as CD9, CD81, CD63, HSP70, Alix, and TSG1016,7, can be measured by Western blotting.
Mammary basal cells have the ability to generate mammary glands when implanted into fat pads, while luminal cells cannot8,9,10. Thus, mammary basal cells are also referred to as mammary repopulating units. By using the model of mammary basal and luminal cells, the ability of EVs/exosomes to transfer cell characteristics between different cell populations can be examined. This work demonstrates the method of transferring gland-forming ability from mammary basal epithelial cells to mammary luminal epithelial cells by using EVs/exosomes derived from mammary basal epithelial cells. Luminal mammary epithelial cells acquired basal cell properties following the ingestion of EVs/exosomes secreted from basal cells and can then form mammary glands4.

<table>
	<tbody>
		<tr>
			<td>MCDB 170&#160;</td>
			<td>USBiological</td>
			<td>M2162</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>Thermo</td>
			<td>1250062</td>
			<td></td>
		</tr>
		<tr>
			<td>Optima L-100K ultracentrifuge</td>
			<td>Beckman</td>
			<td>393253</td>
			<td></td>
		</tr>
		<tr>
			<td>SW28 Ti Rotor</td>
			<td>Beckman</td>
			<td>342204</td>
			<td></td>
		</tr>
		<tr>
			<td>SW41 Rotor</td>
			<td>Beckman</td>
			<td>331306</td>
			<td></td>
		</tr>
		<tr>
			<td>NANOSIGHT LM10</td>
			<td>Malvern</td>
			<td>NANOSIGHT LM10</td>
			<td>for nanoparticle tracking analysis (NTA)</td>
		</tr>
		<tr>
			<td>Optiprep&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>D1556</td>
			<td>60% (w/v) solution of iodixanol in water (sterile).</td>
		</tr>
		<tr>
			<td>CD81 antibody</td>
			<td>GeneTex</td>
			<td>GTX101766</td>
			<td>1:1000 in 5% w/v nonfat dry milk, 1X TBS, 0.1% Tween 20 at 4&#176;C, overnight&#160;</td>
		</tr>
		<tr>
			<td>CD9 antibody</td>
			<td>GeneTex</td>
			<td>GTX100912</td>
			<td>1:1000 in 5% w/v nonfat dry milk, 1X TBS, 0.1% Tween 20 at 4&#176;C, overnight&#160;</td>
		</tr>
		<tr>
			<td>CD63 antibody</td>
			<td>Abcam</td>
			<td>Ab59479</td>
			<td>1:1000 in 5% w/v nonfat dry milk, 1X TBS, 0.1% Tween 20 at 4&#176;C, overnight&#160;</td>
		</tr>
		<tr>
			<td>TSG101 antibody</td>
			<td>GeneTex</td>
			<td>GTX118736</td>
			<td>1:1000 in 5% w/v nonfat dry milk, 1X TBS, 0.1% Tween 20 at 4&#176;C, overnight&#160;</td>
		</tr>
		<tr>
			<td>GAPDH</td>
			<td>GeneTex</td>
			<td>GTX100118</td>
			<td>1:6000 in 5% w/v nonfat dry milk, 1X TBS, 0.1% Tween 20 at 4&#176;C, overnight&#160;</td>
		</tr>
		<tr>
			<td>CFSE (carboxyfluorescein succinimidyl diacetate ester)</td>
			<td>Thermo</td>
			<td>V12883</td>
			<td></td>
		</tr>
		<tr>
			<td>FACSCalibur</td>
			<td>BD Biosciences</td>
			<td></td>
			<td>fluorescence cell analyzer</td>
		</tr>
		<tr>
			<td>collagenase Type IV&#160;</td>
			<td>Thermo</td>
			<td>17104019</td>
			<td></td>
		</tr>
		<tr>
			<td>trypsin</td>
			<td>Thermo</td>
			<td>27250018</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;ITS</td>
			<td>Sigma-Aldrich</td>
			<td>I3146</td>
			<td>a mixture of recombinant human insulin, human transferrin, and sodium selenite</td>
		</tr>
		<tr>
			<td>accutase</td>
			<td>ebioscience</td>
			<td>00-4555-56</td>
			<td>a natural enzyme mixture with proteolytic and collagenolytic enzyme activity</td>
		</tr>
		<tr>
			<td>dispase&#160;</td>
			<td>STEMCELL</td>
			<td>7913</td>
			<td>5 mg/ml = 5 U/ml</td>
		</tr>
		<tr>
			<td>anti-CD49f antibody</td>
			<td>Biolegend</td>
			<td>313611</td>
			<td>1:50</td>
		</tr>
		<tr>
			<td>anti-EpCAM antibody</td>
			<td>Biolegend</td>
			<td>118213</td>
			<td>1:200</td>
		</tr>
		<tr>
			<td>FACSAria</td>
			<td>BD Biosciences</td>
			<td></td>
			<td>cell sorter</td>
		</tr>
		<tr>
			<td>carmine alum</td>
			<td>Sigma-Aldrich</td>
			<td>C1022</td>
			<td></td>
		</tr>
		<tr>
			<td>human mammary epithelial cells (HMLE cells, NAMECs)</td>
			<td></td>
			<td></td>
			<td>gifts from Dr. Robert Weinberg</td>
		</tr>
		<tr>
			<td>permount</td>
			<td>Thermo Fisher Scientific&#160;</td>
			<td>SP15-500</td>
			<td></td>
		</tr>
		<tr>
			<td>sodium bicarbonate</td>
			<td>Zymeset&#160;</td>
			<td>BSB101</td>
			<td></td>
		</tr>
		<tr>
			<td>EGF</td>
			<td>Peprotech</td>
			<td>AF-100-015</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrocoritisone</td>
			<td>Sigma-Aldrich</td>
			<td>SI-H0888</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>SI-I9278</td>
			<td></td>
		</tr>
		<tr>
			<td>BPE (bovine pituitary extract)</td>
			<td>Hammod Cell Tech&#160;</td>
			<td>1078-NZ</td>
			<td></td>
		</tr>
		<tr>
			<td>GW627368X&#160;</td>
			<td>Cayman</td>
			<td>10009162</td>
			<td></td>
		</tr>
		<tr>
			<td>15-cm culture dish</td>
			<td>Falcon&#160;</td>
			<td>353025</td>
			<td></td>
		</tr>
		<tr>
			<td>table-top centrifuge</td>
			<td>Eppendrof&#160;</td>
			<td>Centrifuge 3415R</td>
			<td></td>
		</tr>
		<tr>
			<td>ultracentrifuge tube</td>
			<td>Beckman</td>
			<td>344058</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS (Phosphate-buffered saline)&#160;</td>
			<td>Corning</td>
			<td>46-013-CM</td>
			<td></td>
		</tr>
		<tr>
			<td>BCA Protein Assay</td>
			<td>Thermo Fisher Scientific&#160;</td>
			<td>23228</td>
			<td></td>
		</tr>
		<tr>
			<td>Transmission Electron Microscopy</td>
			<td>Hitachi</td>
			<td>HT7700</td>
			<td></td>
		</tr>
		<tr>
			<td>gelatin&#160;</td>
			<td>STEMCELL</td>
			<td>7903</td>
			<td></td>
		</tr>
		<tr>
			<td>10-cm culture dish</td>
			<td>Falcon&#160;</td>
			<td>353003</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well culture dish</td>
			<td>Corning</td>
			<td>3516</td>
			<td></td>
		</tr>
		<tr>
			<td>female C57BL/6 mice</td>
			<td>NLAC (National Laboratory Animal Center</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>FBS (Fetal Bovine Serum)</td>
			<td>BioWest&#160;</td>
			<td>S01520</td>
			<td></td>
		</tr>
		<tr>
			<td>gentamycin</td>
			<td>Thermo Fisher Scientific&#160;</td>
			<td>15710072</td>
			<td></td>
		</tr>
		<tr>
			<td>Pen/Strep</td>
			<td>Corning</td>
			<td>30-002-Cl</td>
			<td></td>
		</tr>
		<tr>
			<td>DNase I</td>
			<td>5PRIMER</td>
			<td>2500120</td>
			<td></td>
		</tr>
		<tr>
			<td>isofluorane&#160;</td>
			<td>Halocarbon</td>
			<td>NPC12164-002-25</td>
			<td></td>
		</tr>
		<tr>
			<td>formaldehyde</td>
			<td>MACRON</td>
			<td>H121-08</td>
			<td></td>
		</tr>
		<tr>
			<td>EtOH (Ethanol)</td>
			<td>J.T. Baker</td>
			<td>800605</td>
			<td></td>
		</tr>
		<tr>
			<td>glacial acetic acid</td>
			<td>Panreac</td>
			<td>131008.1611</td>
			<td></td>
		</tr>
		<tr>
			<td>aluminum potassium sulfate</td>
			<td>Sigma-Aldrich</td>
			<td>12625</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylene&#160;</td>
			<td>Leica</td>
			<td>3803665</td>
			<td></td>
		</tr>
		<tr>
			<td>0.22 &#956;m membranes</td>
			<td>Merck Millipore</td>
			<td>Millex-GP</td>
			<td></td>
		</tr>
		<tr>
			<td>AUTOCLIP Wound Clips, 9 mm</td>
			<td>BD Biosciences</td>
			<td>427631</td>
			<td></td>
		</tr>
		<tr>
			<td>AUTOCLIP Wound Clip Applier</td>
			<td>BD Biosciences</td>
			<td>427630</td>
			<td></td>
		</tr>
		<tr>
			<td>CellMask&#8482; Deep Red</td>
			<td>Thermo Fisher Scientific&#160;</td>
			<td>C10046</td>
			<td>plasma membrane stain</td>
		</tr>
	</tbody>
</table>

transfer of mammary gland-forming ability between mammary basal epithelial cells and mammary luminal cells via extracellular vesicles/exosomes

Cells can communicate via exosomes, ~100-nm extracellular vesicles (EVs) that contain proteins, lipids, and nucleic acids. Non-adherent/mesenchymal mammary epithelial cell (NAMEC)-derived extracellular vesicles can be isolated from NAMEC medium via differential ultracentrifugation. Based on their density, EVs can be purified via ultracentrifugation at 110,000 x g. The EV preparation from ultracentrifugation can be further separated using a continuous density gradient to prevent contamination with soluble proteins. The purified EVs can then be further evaluated using nanoparticle-tracking analysis, which measures the size and number of vesicles in the preparation. The extracellular vesicles with a size ranging from 50 to 150 nm are exosomes. The NAMEC-derived EVs/exosomes can be ingested by mammary epithelial cells, which can be measured by flow cytometry and confocal microscopy. Some mammary stem cell properties (e.g., mammary gland-forming ability) can be transferred from the stem-like NAMECs to mammary epithelial cells via the NAMEC-derived EVs/exosomes. Isolated primary EpCAMhi/CD49flo luminal mammary epithelial cells cannot form mammary glands after being transplanted into mouse fat pads, while EpCAMlo/CD49fhi basal mammary epithelial cells form mammary glands after transplantation. Uptake of NAMEC-derived EVs/exosomes by EpCAMhi/CD49flo luminal mammary epithelial cells allows them to generate mammary glands after being transplanted into fat pads. The EVs/exosomes derived from stem-like mammary epithelial cells transfer mammary gland-forming ability to EpCAMhi/CD49flo luminal mammary epithelial cells.

Since it has been shown that blocking PGE2/EP4 signaling triggers EV/exosome release from mammary basal-like stem cells4, this work presents a method of isolating the induced EVs/exosomes from mammary epithelial basal cell (NAMEC) culture. Since NAMECs are cultured in serum-free medium, there are no pre-existing EVs/exosomes derived from serum13. For cells cultured in serum-containing medium, pre-existing exosomes in th...

Watch this Scientific Journal Video about Transfer of Mammary Gland-forming Ability Between Mammary Basal Epithelial Cells and Mammary Luminal Cells via Extracellular Vesicles/Exosomes at JoVE.com

Transfer of Mammary Gland-forming Ability Between Mammary Basal Epithelial Cells and Mammary Luminal Cells via Extracellular Vesicles/Exosomes

This protocol describes methods for purifying, quantitating, and characterizing extracellular vesicles (EVs)/exosomes from non-adherent/mesenchymal mammary epithelial cells and for using them to transfer mammary gland-forming ability to luminal mammary epithelial cells. EVs/exosomes derived from stem-like mammary epithelial cells can transfer this cell property to cells that ingest the EVs/exosomes.

Cells can communicate via exosomes, ~100-nm extracellular vesicles (EVs) that contain proteins, lipids, and nucleic acids. Non-adherent/mesenchymal ...

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JoVE Journal

Developmental Biology

11.6K Views.  National Health Research Institutes. This protocol describes methods for purifying, quantitating, and characterizing extracellular vesicles (EVs)/exosomes from non-adherent/mesenchymal mammary epithelial cells and for using them to transfer mammary gland-forming ability to luminal mammary epithelial cells. EVs/exosomes derived from stem-like mammary epithelial cells can transfer this cell property to cells that ingest the EVs/exosomes.

Video: Transfer of Mammary Gland-forming Ability Between Mammary Basal Epithelial Cells and Mammary Luminal Cells via Extracellular Vesicles/Exosomes

Cultivate Primary Nasal Epithelial Cells from Children and Reprogram into Induced Pluripotent Stem Cells

Nasal epithelial cells (NECs) are the part of the airways that respond to air pollutants and are the first cells infected with respiratory viruses. They are also involved in many airway diseases through their innate immune response and interaction with immune and airway stromal cells. NECs are of particular interest for studies in children due to their accessibility during clinical visits. Human induced pluripotent stem cells (iPSCs) have been generated from multiple cell types and are a powerful tool for modeling human development and disease, as well as for their potential applications in regenerative medicine. This is the first protocol to lay out methods for successful generation of iPSCs from NECs derived from pediatric participants for research purposes. It describes how to obtain nasal epithelial cells from children, how to generate primary NEC cultures from these samples, and how to reprogram primary NECs into well-characterized iPSCs. Nasal mucosa samples are useful in epidemiological studies related to the effects of air pollution in children, and provide an important tool for studying airway disease. Primary nasal cells and iPSCs derived from them can be a tool for providing unlimited material for patient-specific research in diverse areas of airway epithelial biology, including asthma and COPD research.

This publication demonstrates methods for successful sampling and culture of nasal epithelial mucosa from children, and reprogramming these cells to induced Pluripotent Stem Cells (iPSCs).

Nasal epithelial cells (NECs) are the part of the airways that respond to air pollutants and are the first cells infected with respiratory viruses. They ...

Induction of Mesenchymal-Epithelial Transitions in Sarcoma Cells

Phenotypic plasticity refers to a phenomenon in which cells transiently gain traits of another lineage. During carcinoma progression, phenotypic plasticity drives invasion, dissemination and metastasis. Indeed, while most of the studies of phenotypic plasticity have been in the context of epithelial-derived carcinomas, it turns out sarcomas, which are mesenchymal in origin, also exhibit phenotypic plasticity, with a subset of sarcomas undergoing a phenomenon that resembles a mesenchymal-epithelial transition (MET). Here, we developed a method comprising the miR-200 family and grainyhead-like 2 (GRHL2) to mimic this MET-like phenomenon observed in sarcoma patient samples.We sequentially express GRHL2 and the miR-200 family using cell transduction and transfection, respectively, to better understand the molecular underpinnings of these phenotypic transitions in sarcoma cells. Sarcoma cells expressing miR-200s and GRHL2 demonstrated enhanced epithelial characteristics in cell morphology and alteration of epithelial and mesenchymal biomarkers. Future studies using these methods can be used to better understand the phenotypic consequences of MET-like processes on sarcoma cells, such as migration, invasion, metastatic propensity, and therapy resistance.

We present here a cell culture method for inducing mesenchymal-epithelial transitions (MET) in sarcoma cells based on combined ectopic expression of microRNA-200 family members and grainyhead-like 2 (GRHL2). This method is suitable for better understanding the biological impact of phenotypic plasticity on cancer aggressiveness and treatments.

Phenotypic plasticity refers to a phenomenon in which cells transiently gain traits of another lineage. During carcinoma progression, phenotypic ...

Analysis of Protein-protein Interactions and Co-localization Between Components of Gap, Tight, and Adherens Junctions in Murine Mammary Glands

Cell-cell interactions play a pivotal role in preserving tissue integrity and the barrier between the different compartments of the mammary gland. These interactions are provided by junctional proteins that form nexuses between adjacent cells. Junctional protein mislocalization and reduced physical associations with their binding partners can result in the loss of function and, consequently, to organ dysfunction. Thus, identifying protein localization and protein-protein interactions (PPIs) in normal and disease-related tissues is essential to finding new evidences and mechanisms leading to the progression of diseases or alterations in developmental status. This manuscript presents a two-step method to evaluate PPIs in murine mammary glands. In protocol section 1, a method to perform co-immunofluorescence (co-IF) using antibodies raised against the proteins of interest, followed by secondary antibodies labeled with fluorochromes, is described. Although co-IF allows for the demonstration of the proximity of the proteins, it does make it possible to study their physical interactions. Therefore, a detailed protocol for co-immunoprecipitation (co-IP) is provided in protocol section 2. This method is used to determine the physical interactions between proteins, without confirming whether these interactions are direct or indirect. In the last few years, co-IF and co-IP techniques have demonstrated that certain components of intercellular junctions co-localize and interact together, creating stage-dependent junctional nexuses that vary during mammary gland development.

Intercellular junctions are requisites for mammary gland stage-specific functions and development. This manuscript provides a detailed protocol for the study of protein-protein interactions (PPIs) and co-localization using murine mammary glands. These techniques allow for the investigation of the dynamics of the physical association between intercellular junctions at different developmental stages.

Cell-cell interactions play a pivotal role in preserving tissue integrity and the barrier between the different compartments of the mammary gland. These ...

Quantifying Branching Density in Rat Mammary Gland Whole-mounts Using the Sholl Analysis Method

An increasing number of studies are utilizing the rodent mammary gland as an endpoint for assessing the developmental toxicity of a chemical exposure. The effects these exposures have on mammary gland development are typically evaluated using either basic dimensional measurements or by scoring morphological characteristics. However, the broad range of methods for interpreting developmental changes could lead to inconsistent translations across laboratories. A common method of assessment is needed so that proper interpretations can be formed from data being compared across studies. The present study describes the application of the Sholl analysis method to quantify mammary gland branching characteristics. The Sholl method was originally developed for use in quantifying neuronal dendritic patterns. By using ImageJ, an open-source image analysis software package, and a plugin developed for this analysis, the mammary gland branching density and the complexity of a mammary gland from a peripubertal female rat were determined. The methods described here will enable the use of the Sholl analysis as an effective tool for quantifying an important characteristic of mammary gland development.

Mammary gland development in the rodent has typically been evaluated using descriptive assessments or by measuring basic physical attributes. Branching density is an indicator of mammary development that is difficult to quantify objectively. This protocol describes a reliable method for the quantitative assessment of mammary gland branching characteristics.

An increasing number of studies are utilizing the rodent mammary gland as an endpoint for assessing the developmental toxicity of a chemical exposure. The ...

A Novel Mammary Fat Pad Transplantation Technique to Visualize the Vessel Generation of Vascular Endothelial Stem Cells

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper vessel development and homeostasis. However, studies on endothelial lineage hierarchy remain elusive due to the lack of tools to gain access as well as to directly evaluate their behavior in vivo. To address this shortcoming, a new tissue model to study angiogenesis using the mammary fat pad has been developed. The mammary gland develops mostly in the postnatal stages, including puberty and pregnancy, during which robust epithelium proliferation is accompanied by extensive vascular remodeling. Mammary fat pads provide space, matrix, and rich angiogenic stimuli from the growing mammary epithelium. Furthermore, mammary fat pads are located outside the peritoneal cavity, making them an easily accessible grafting site for assessing the angiogenic potential of exogenous cells. This work also describes an efficient tracing approach using fluorescent reporter mice to specifically label the targeted population of vascular endothelial stem cells (VESCs) in vivo. This lineage tracing method, coupled with subsequent tissue whole-mount microscopy, enable the direct visualization of targeted cells and their descendants, through which the proliferation capability can be quantified and the differentiation commitment can be fate-mapped. Using these methods, a population of bipotent protein C receptor (Procr) expressing VESCs has recently been identified in multiple vascular systems. Procr+ VESCs, giving rise to both new ECs and pericytes, actively contribute to angiogenesis during development, homeostasis, and injury repair. Overall, this manuscript describes a new mammary fat pad transplantation and in vivo lineage tracing techniques that can be used to evaluate the stem cell properties of VESCs.

This work demonstrates a novel approach to assess the proliferation, differentiation, and vessel-forming potential of vascular endothelial stem cells (VESCs) through mammary fat pad transplantation followed by whole-mount tissue preparation for microscopic observation. A lineage tracing strategy to investigate the behavior of VESCs in vivo is also presented.

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper ...

Horizontal Whole Mount: A Novel Processing and Imaging Protocol for Thick, Three-dimensional Tissue Cross-sections of Skin

Processing a tissue of interest to generate a microscopic image that supports a scientific argument can be challenging. The acquisition of high-quality microscopic images is not entirely dependent upon the quality of the microscope, but also upon the methods of tissue processing, which often involve multiple critical actions or steps. Furthermore, mesenchymal cell types in the skin and other tissues represent a new challenge for tissue preparation and imaging. Here, we present a complete process, from tissue harvest to microscopy. Our technique, called &#34;horizontal whole mount,&#34; is one that novices can quickly become proficient in and that allows for antigen preservation and detection in 60-300 &#181;m-thick sections cut with a cryostat. Sections of this thickness provide enhanced visualization of tissue microarchitecture in a three-dimensional environment. In addition, the protocol preserves mesenchymal cells in a manner that enhances image quality when compared to standard cryostat or paraffin sections, thereby increasing the efficacy and reliability of immunostaining. We believe that this protocol will benefit all laboratories that visualize skin, and possibly other tissues and organs.

This work presents a novel processing and imaging protocol for thick, three-dimensional tissue cross-section analysis that enables the full exploitation of confocal imaging modalities. This protocol preserves antigenicity and represents a robust system to analyze skin histology and potentially other tissue types.

Processing a tissue of interest to generate a microscopic image that supports a scientific argument can be challenging. The acquisition of high-quality ...

Single-cell Photoconversion in Living Intact Zebrafish

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause disease when disrupted. Scientists have developed clever techniques to investigate characteristics and natural dynamics of these cells within intact tissue by expressing fluorescent proteins in subsets of cells. However, at times, experiments require more selected visualization of cells within the tissue, sometimes at the single-cell or population-of-cells manner. To achieve this and visualize single cells within a population of cells, scientists have utilized single-cell photoconversion of fluorescent proteins. To demonstrate this technique, we show here how to direct UV light to an Eos-expressing cell of interest in an intact, living zebrafish. We then image those photoconverted Eos+ cells 24 h later to determine how they changed in the tissue. We describe two techniques: single cell photoconversion and photoconversions of populations of cell. These techniques can be used to visualize cell-cell interactions, cell-fate and differentiation, and cell migrations, making it a technique that is applicable in numerous biological questions.

Here, we present a protocol to show how cell photoconversion is achieved through UV exposure to specific areas expressing the fluorescent protein, Eos, in living animals.

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause ...

Identification of Skeletal Muscle Satellite Cells by Immunofluorescence with Pax7 and Laminin Antibodies

Immunofluorescence is an effective method that helps to identify different cell types on tissue sections. In order to study the desired cell population, antibodies for specific cell markers are applied on tissue sections. In adult skeletal muscle, satellite cells (SCs) are stem cells that contribute to muscle repair and regeneration. Therefore, it is important to visualize and trace the satellite cell population under different physiological conditions. In resting skeletal muscle, SCs reside between the basal lamina and myofiber plasma membrane. A commonly used marker for identifying SCs on the myofibers or in cell culture is the paired box protein Pax7. In this article, an optimized Pax7 immunofluorescence protocol on skeletal muscle sections is presented that minimizes non-specific staining and background. Another antibody that recognizes a protein (laminin) of the basal lamina was also added to help identify SCs. Similar protocols can also be used to perform double or triple labeling with Pax7 and antibodies for additional proteins of interest.

The precise identification of satellite cells is essential for studying their functions under various physiological and pathological conditions. This article presents a protocol to identify satellite cells on adult skeletal muscle sections by immunofluorescence-based staining.

Immunofluorescence is an effective method that helps to identify different cell types on tissue sections. In order to study the desired cell population, ...

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and chromosome dynamics. While the germline is an excellent model, the analysis is often two dimensional due to the time and labor required for three-dimensional analysis. Major readouts in such studies are the number/position of nuclei and protein distribution within the germline. Here, we present a method to perform automated analysis of the germline using confocal microscopy and computational approaches to determine the number and position of nuclei in each region of the germline. Our method also analyzes germline protein distribution that enables the three-dimensional examination of protein expression in different genetic backgrounds. Further, our study shows variations in cytoskeletal architecture in distinct regions of the germline that may accommodate specific spatial developmental requirements. Finally, our method enables automated counting of the sperm in the spermatheca of each germline. Taken together, our method enables rapid and reproducible phenotypic analysis of the C. elegans germline.

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

We present an automated method for three-dimensional reconstruction of the Caenorhabditis elegans germline. Our method determines the number and position of each nucleus within the germline and analyses germline protein distribution and cytoskeletal structure.

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and ...

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give rise to multiple cell types, including the myotome (MYO), sclerotome (SCL), dermatome (D), and syndetome (SYN), which in turn develop into skeletal muscle, axial skeleton, dorsal dermis, and axial tendon/ligament, respectively. Therefore, the generation of SMs and their derivatives from human induced pluripotent stem cells (iPSCs) is critical to obtain pluripotent stem cells (PSCs) for application in regenerative medicine and for disease research in the field of orthopedic surgery. Although the induction protocols for MYO and SCL from PSCs have been previously reported by several researchers, no study has yet demonstrated the induction of SYN and D from iPSCs. Therefore, efficient induction of fully competent SMs remains a major challenge. Here, we recapitulate human SM patterning with human iPSCs in vitro by mimicking the signaling environment during chick/mouse SM development, and report on methods of systematic induction of SM derivatives (MYO, SCL, D, and SYN) from human iPSCs under chemically defined conditions through the presomitic mesoderm (PSM) and SM states. Knowledge regarding chick/mouse&#160;SM development was successfully applied to the induction of SMs with human iPSCs. This method could be a novel tool for studying human somitogenesis and patterning without the use of embryos and for cell-based therapy and disease modeling.

We present here a protocol for the differentiation of human induced pluripotent stem cells into each somite derivative (myotome, sclerotome, dermatome, and syndetome) in chemically defined conditions, which has applications in future disease modeling and cell-based therapies in orthopedic surgery.

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...