Name: isolation of micrornas from tick ex vivo salivary gland cultures and extracellular vesicles
Uploaded: 2022-04-06T14:00:00
Description: Watch this Scientific Journal Video about Isolation of microRNAs from Tick Ex Vivo Salivary Gland Cultures and Extracellular Vesicles at JoVE.com

We are greatly appreciative for the assistance from the Cattle Fever tick Laboratory in Edinburg, Texas. We would like to thank Michael Moses, Jason Tidwell, James Hellums, Cesario Agado, and Homer Vasquez. We would also like to acknowledge the assistance of Sarah Sharpton, Elizabeth Lohstroh, Amy Filip, Kelsey Johnson, Kelli Kochcan, Andrew Hillhouse, Charluz Arocho Rosario, and Stephanie Guzman Valencia throughout the project. We would like to thank the Texas A&#38;M Aggie Women in Entomology (AWE) Writing Group for their help and advice during the writing of this manuscript. The following reagents were provided by Centers for Disease Control and Prevention for distribution by BEI Resources, NIAID, NIH: Ixodes scapularis Adult (Live), NR-42510. Female I. scapularis ticks were also received from the Tick Rearing Facility at Oklahoma State University. This project was funded by Texas A&#38;M University T3: triads for transformation grant and the cooperative agreement #58-3094-1-003 by the USDA-ARS to AOC.

All animal experiments were performed following animal usage protocol (AUP#2020-0026) approved by the institutional animal care and use committee (AICUC) at Texas A&#38;M University. The tick species, Ixodes scapularis&#160;and Rhipicephalus (Boophilus) microplus, and New Zealand Male White Rabbits, 42-72 days of age, were used for the present study. I. scapularis was received from the Center for Disease Control (CDC) and Oklahoma State University, certified as pathogen-free.&#160;R. microp...

The current protocol provides a detailed methodology for extracting miRNA from salivary glands and EVs. However, there are important considerations, all of which are detailed in the notes for each section of this protocol. The capsule and mesh netting must be secured during tick feeding to prevent ticks from escaping. The capsule preparation and placement are described in Koga et al.40. Several replicates of the tick dissections need to be done if an unsuitable sample is discarded. Additionally, s...

Ticks are ectoparasites that vector many pathogens to wildlife, livestock, humans, and their pets1,2. Tick feeding results in significant economic loss by causing damage to hide, reducing weight and milk production due to severe anemia, and the transmission of potentially deadly disease-causing pathogens1,3,4,5. Current control practices for managing tick populations are focused on the use of acaricides. Nevertheless, the continuous emergence of acaricide resistance in ticks parasitizing livestock5,6, the increased incidence of tick bites7, and pathogen transmission within residential areas8,9, have led to a need for unique tick control alternatives.
The tick salivary glands are essential organs that ensure a tick&#39;s biological success. They are formed by different acinus types (I, II, III, and IV) with various physiological functions. The salivary glands are responsible for osmoregulation, both off and on the host, by returning water excess and iron content to the host via salivation2,10. Type I acini are also involved in the uptake of water from the atmosphere by the secretion of hygroscopic saliva10,11. Salivary effector proteins, such as cement and cystatins, are produced within secretory cells in type II and III acini10,12. Type I acini do not affect tick feeding, indicating that the bloodmeal intake does not trigger morphological and physiological changes in these acini type13,14. On the other hand, Acini type II and III are activated during feeding and present very little activity pre-attachment. Thus, feeding is necessary to trigger the enlargement of the secretory cells within type II acini and the production of bioactive compounds. Type III acini are reduced in size during feeding due to the secretion within the secretory granules12.
The salivary glands are also the site of pathogen infection in the tick and route of transmission. During feeding, ticks secrete several compounds with pharmaceutical effects that are needed for successful completion of the bloodmeal10,15,16. These compounds have anti-inflammatory, immunosuppressive, and vasodilatory properties10,15,17. Recent studies have shown that extracellular vesicles (EVs) derived from tick salivary glands harbor several of these compounds, inducing anti-inflammatory and immuno-modulatory effects18,19,20. &#34;Extracellular vesicles&#34; is an umbrella term used to describe vesicles classified as exosomes and microvesicles based on their size and biogenesis. Overall, EVs are lipid blebs with bilayer membranes that are ~40 nm-1 &#181;m in size21; generally, exosomes are described as being 40-150 nm in size, whereas microvesicles are between 150 nm-1 &#181;m in size21,22,23. However, the size is not indicative of the EVs biogenesis pathway22.
The biogenesis of exosomes starts with the sequential invagination of the plasma membrane. This invagination leads to the formation of multivesicular bodies and finally results in the deformation of the vesicular membrane by the action of ESCRT complexes or sphingomyelinases (sMases)24,25. The exosomes can either be lysed within the lysosomes to maintain cellular homeostasis or exit via vesicular fusion to the plasma membrane to deliver cellular constituents to the recipient cells21,24. On the other hand, microvesicles are formed by the action of flopasses and flipasses, changing the conformation of lipids in the plasma membrane26. EVs are essential for cell-to-cell communication, serving as a transport system for intracellular cargo, such as lipids, proteins, nucleic acids, and microRNAs (miRNAs)21,27,28. Once transported, these vesicles deliver their cargo into the cytoplasm of the recipient cells, generating phenotypic changes in the receiving cell22,29. Due to the importance of extracellular vesicles in tick feeding and the manipulation of host immune and wound healing responses18,20, the cargo within extracellular vesicles presents potential targets for the development of anti-tick therapeutics and a unique mechanism to disrupt tick feeding. This includes miRNAs within tick salivary glands and salivary gland-derived extracellular vesicles.
miRNAs are short non-coding sequences, ~18-22 nucleotide (nt) in length, that can post-transcriptionally regulate, degrade, or silence mRNA sequences30,31. During transcription, the pri-miRNAs are cleaved by Dicer (RNA polymerase III) to form a distinctive hairpin-like structure, becoming a pre-miRNA. The pre-miRNA is cut once again by Drosha (RNA polymerase III) to form a mature miRNA duplex. The mature sequence becomes integrated into the RNA-induced silencing complex (RISC) complementary to the mRNA sequence, causing translation repression or mRNA degradation28,30,32. During host feeding, miRNAs within the tick saliva can modulate host gene expression to suppress immune responses and enhance pathogen transmission33,34,35,36,37. Although extensive studies on EVs and miRNAs exist, their roles during feeding at the tick-host interface are still poorly understood. Optimizing protocols that can easily result in the isolation and purification of high-quality miRNAs is crucial for advancing our knowledge on these topics.
Multiple options can be utilized to isolate EVs, such as ultracentrifugation, exosome precipitation, polymer precipitation, immunoaffinity chromatography, and size-based exclusion techniques38. However, these techniques cannot distinguish between exosomes or microvesicles. Thus, as mentioned previously, EV is used as an umbrella term when isolating EVs from different samples. The vesicles isolated in the experiments described herein represent a mixture of vesicles derived from different biogenesis pathways. Further purification of a specific population of extracellular vesicles can be achieved by immunoprecipitation using beads coated with antibodies against markers (i.e., exosomal markers, tumor markers) unique to the vesicle population of interest39,40. miRNAs can also be extracted via different commercially available isolation kits7,41,42.
The objective of this project was to develop a protocol that combines commonly applied methods to isolate EVs and extract miRNA from both EVs and fed-tick salivary glands. Because the secretion of bioactive compounds is activated by feeding12, ticks should be allowed to feed to identify miRNAs that may be important for manipulating host immune and wound healing responses. The present protocol requires a small number of ticks (20 ticks) to isolate EVs and their respective miRNAs, compared to other previously described studies that required 2000 ticks43. Further, it avoids the contamination of salivary secretions with pilocarpine44, which could influence experiments studying the effect of EVs and their miRNAs on host cells.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.22 &#181;m syringe filter</td>
			<td>GenClone</td>
			<td>25-240</td>
			<td></td>
		</tr>
		<tr>
			<td>1 &#181;m nylon syringe filter</td>
			<td>Tisch Scientific</td>
			<td>283129028</td>
			<td></td>
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		<tr>
			<td>1 inch black adhesive</td>
			<td>Amazon</td>
			<td>B00FQ937NM</td>
			<td>Capsule</td>
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		<tr>
			<td>10 mL needeless syringe</td>
			<td>Exelint</td>
			<td>26265</td>
			<td></td>
		</tr>
		<tr>
			<td>3&#39; and 5&#39; Adapters</td>
			<td>Illumina</td>
			<td>20024906</td>
			<td>NEXTFLEX Small RNA-Seq Kit</td>
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		<tr>
			<td>4 mm vannas scissors</td>
			<td>Fine Science Tools</td>
			<td>15000-08</td>
			<td></td>
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			<td>4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid</td>
			<td>Sigma-Aldrich</td>
			<td>1.1523</td>
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		<tr>
			<td>70Ti rotor</td>
			<td>Beckman Coulter</td>
			<td>337922</td>
			<td></td>
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			<td>Amphotericin</td>
			<td>Corning</td>
			<td>30-003-CF</td>
			<td></td>
		</tr>
		<tr>
			<td>Beads</td>
			<td>Illumina</td>
			<td>20024906</td>
			<td>NEXTFLEX Small RNA-Seq Kit</td>
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			<td>Bioanalyzer</td>
			<td>Agilent</td>
			<td>G2939BA</td>
			<td></td>
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			<td>Bioanalyzer kit</td>
			<td>Agilent</td>
			<td>5067-1513</td>
			<td></td>
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		<tr>
			<td>Centrifuge 5425</td>
			<td>Eppendorf</td>
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			<td></td>
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			<td>Chloroform</td>
			<td>Macron</td>
			<td>UN1888</td>
			<td></td>
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		<tr>
			<td>Cyverse Discovery Enviornment</td>
			<td></td>
			<td></td>
			<td>https://cyverse.org/discovery-environment</td>
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		<tr>
			<td>Dissecting microscope</td>
			<td>Nikon</td>
			<td>SMZ745</td>
			<td></td>
		</tr>
		<tr>
			<td>Double-sideded carpet tape</td>
			<td>amazon</td>
			<td>&#8206;286373</td>
			<td></td>
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		<tr>
			<td>Falcon Tubes, 50 mL</td>
			<td>VWR</td>
			<td>21008-940</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Gibco</td>
			<td>FBS-02-0050</td>
			<td></td>
		</tr>
		<tr>
			<td>fine forceps</td>
			<td>Excelta</td>
			<td>5-S-SE</td>
			<td></td>
		</tr>
		<tr>
			<td>Foamies, 2 mm</td>
			<td>Amazon</td>
			<td>B004M5QGBQ</td>
			<td>Capsule</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Phoenix Pharmaceuticals manfactured</td>
			<td>193.33165.3</td>
			<td></td>
		</tr>
		<tr>
			<td>Ixodes scaplaris</td>
			<td>CDC, Oklahoma State University</td>
			<td></td>
			<td></td>
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		<tr>
			<td>L15C300 medium</td>
			<td>In-lab</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>lipoprotein-cholesterol concentrate</td>
			<td>MPI</td>
			<td>02191476-CF</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope slide</td>
			<td>VWR</td>
			<td>10118-596</td>
			<td></td>
		</tr>
		<tr>
			<td>miRDeep2</td>
			<td></td>
			<td></td>
			<td>https://github.com/rajewsky-lab/mirdeep2</td>
		</tr>
		<tr>
			<td>M-MuLV Reverse Transcriptase</td>
			<td>Illumina</td>
			<td>20024906</td>
			<td>NEXTFLEX Small RNA-Seq Kit</td>
		</tr>
		<tr>
			<td>molecular grade ethanol</td>
			<td>Fischer Bioreagents</td>
			<td>UN1170</td>
			<td></td>
		</tr>
		<tr>
			<td>multi-well 24 well tissue culture treated plate</td>
			<td>Corning</td>
			<td>353047</td>
			<td></td>
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		<tr>
			<td>Nanopaticle Tracking Analyzer machine</td>
			<td>Malvern Panalytical</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nanosep with 300K Omega filter</td>
			<td>Pall Corporation</td>
			<td>OD3003C33</td>
			<td></td>
		</tr>
		<tr>
			<td>NEXTFLEX Small RNA-Seq Kit v3</td>
			<td>PerkinElmer</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NextSeq 500/550 High Output Kit (75 cycles)</td>
			<td>Illumina</td>
			<td>20024906</td>
			<td></td>
		</tr>
		<tr>
			<td>Optima XPN 90 Ultracentrifuge</td>
			<td>Beckman Coulter</td>
			<td></td>
			<td></td>
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		<tr>
			<td>Penicillin</td>
			<td>Thermofischer Scientific</td>
			<td>ICN19453780</td>
			<td></td>
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		<tr>
			<td>Pippettes</td>
			<td>Ependorff</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>polycarbonate centrifuge bottle</td>
			<td>Beckman Coulter</td>
			<td>355618</td>
			<td></td>
		</tr>
		<tr>
			<td>Qiagen miRNeasy kit</td>
			<td>Qiagen</td>
			<td>217084</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAzol lysis reagent</td>
			<td>Qiagen</td>
			<td>79306</td>
			<td></td>
		</tr>
		<tr>
			<td>Qubit</td>
			<td>Thermofisher</td>
			<td>Q32880</td>
			<td></td>
		</tr>
		<tr>
			<td>Qubit kit</td>
			<td>Thermofisher</td>
			<td>Q10212</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbits</td>
			<td>Charles River</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Reverse Universal Primer</td>
			<td>Illumina</td>
			<td>20024906</td>
			<td>NEXTFLEX Small RNA-Seq Kit</td>
		</tr>
		<tr>
			<td>Rhipicephalus microplus</td>
			<td>Cattle Fever Tick Research Labratoty</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Rifampicin</td>
			<td>Fischer Bioreagents</td>
			<td>215544</td>
			<td></td>
		</tr>
		<tr>
			<td>RNAlater</td>
			<td>Invitrogen</td>
			<td>833280</td>
			<td></td>
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		<tr>
			<td>RNAse free tubes</td>
			<td>VWR</td>
			<td>87003294</td>
			<td></td>
		</tr>
		<tr>
			<td>RNAse inhibitor</td>
			<td>Thermo Fischer</td>
			<td>11111729</td>
			<td></td>
		</tr>
		<tr>
			<td>RNAse/DNAse free water</td>
			<td>Qiagen</td>
			<td>217084</td>
			<td></td>
		</tr>
		<tr>
			<td>RNeasy Minelute spin column</td>
			<td>Qiagen</td>
			<td>217084</td>
			<td>Qiagen miRNeasy kit</td>
		</tr>
		<tr>
			<td>RPE Buffer</td>
			<td>Qiagen</td>
			<td>217084</td>
			<td>Qiagen miRNeasy kit</td>
		</tr>
		<tr>
			<td>RT Buffer</td>
			<td>Illumina</td>
			<td>20024906</td>
			<td>NEXTFLEX Small RNA-seq kit</td>
		</tr>
		<tr>
			<td>RT Forward Primer</td>
			<td>Illumina</td>
			<td>20024906</td>
			<td>NEXTFLEX Small RNA-seq kit</td>
		</tr>
		<tr>
			<td>RTE Buffer</td>
			<td>Qiagen</td>
			<td>217084</td>
			<td>Qiagen miRNeasy kit</td>
		</tr>
		<tr>
			<td>Sodium bicarbonate</td>
			<td>Sigma-Aldrich</td>
			<td>S6014-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Sorvall ST16</td>
			<td>Thermo Fischer</td>
			<td>75004380</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterilized Gauze sponges</td>
			<td>Covidien</td>
			<td>2187</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterilized PBS</td>
			<td>Sigma</td>
			<td>RNBK0694</td>
			<td></td>
		</tr>
		<tr>
			<td>streptomycin</td>
			<td>thermofischer Scientific</td>
			<td>15240062</td>
			<td></td>
		</tr>
		<tr>
			<td>TapeStation</td>
			<td>Aligent</td>
			<td>G2991BA</td>
			<td></td>
		</tr>
		<tr>
			<td>Tear Mender Instant Fabric and Leather Adhesive</td>
			<td>Amazon</td>
			<td>7.42836E+11</td>
			<td>Capsule</td>
		</tr>
		<tr>
			<td>Tissue Adhesive</td>
			<td>3M VetBond</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Triple Antibiotics</td>
			<td>dechra</td>
			<td>17033-122-75</td>
			<td></td>
		</tr>
		<tr>
			<td>Tryptose phosphate broth</td>
			<td>BD</td>
			<td>BD 260300</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation of micrornas from tick ex vivo salivary gland cultures and extracellular vesicles

Ticks are important ectoparasites that can vector multiple pathogens. The salivary glands of ticks are essential for feeding as their saliva contains many effectors with pharmaceutical properties that can diminish host immune responses and enhance pathogen transmission. One group of such effectors are microRNAs (miRNAs). miRNAs are short non-coding sequences that regulate host gene expression at the tick-host interface and within the organs of the tick. These small RNAs are transported in the tick saliva via extracellular vesicles (EVs), which serve inter-and intracellular communication. Vesicles containing miRNAs have been identified in the saliva of ticks. However, little is known about the roles and profiles of the miRNAs in tick salivary vesicles and glands. Furthermore, the study of vesicles and miRNAs in tick saliva requires tedious procedures to collect tick saliva. This protocol aims to develop and validate a method for isolating miRNAs from purified extracellular vesicles produced by ex vivo organ cultures. The materials and methodology needed to extract miRNAs from extracellular vesicles and tick salivary glands are described herein.

The present protocol provides a detailed methodology to extract miRNAs from salivary glands and EVs. According to the results, this protocol is effective for the isolation of miRNA from adults of two different tick species, I. scapularis and R. microplus, and can potentially be used in other tick species as well. The EVs concentration (particles/mL) was measured via NTA. For R. microplus, each gender and life stage contained three biological replicates measured in three technical repli...

Watch this Scientific Journal Video about Isolation of microRNAs from Tick Ex Vivo Salivary Gland Cultures and Extracellular Vesicles at JoVE.com

Isolation of microRNAs from Tick Ex Vivo Salivary Gland Cultures and Extracellular Vesicles

The present protocol describes the isolation of microRNAs from tick salivary glands and purified extracellular vesicles. This is a universal procedure that combines commonly used reagents and supplies. The method also allows the use of a small number of ticks, resulting in quality microRNAs that can be readily sequenced.

Isolation of microRNAs from Tick Ex Vivo Salivary Gland Cultures and Extracellular Vesicles

Ticks are important ectoparasites that can vector multiple pathogens. The salivary glands of ticks are essential for feeding as their saliva contains many ...

This protocol allows us to use a reduced number of ticks which reduces the cost of colony maintenance and decreases the number of vertebrae animals needed for tick feeding. This protocol requires approximately 20 ticks. However, the main advantage aside from using a small sample size is that this protocol can be applied to multiple tick species and different life stages.Aside from tick dissections or the application towards host-parasite interactions, the effect of pathogen infection on microRNA secretion and the effect of physiological changes on microRNA secreted by ticks. To begin, prepare vesicle-free media by adding fetal bovine serum, tryptose phosphate broth, 10%lipoprotein cholesterol concentrate, 5%sodium bicarbonate, HEPES, and L15C300 medium into a 26.3 milliliter polycarbonate centrifuge bottle and adjust the volume as required. Now ultracentrifuge the medium at 100, 000 times G at four degrees Celsius for 18 hours.Carefully remove the supernatant without disturbing the pellet and transfer the supernatant to a fresh centrifuge tube. Ultracentrifuge the tube once again. Take the supernatant and pass it through a 0.22 micrometer filter to remove contaminants.Pipette the supernatant into a 50 milliliter centrifuge tube and store it at minus 20 degrees Celsius until required or up to three years. Add an appropriate amount of antibiotics to 500 microliters of the vesicle-free medium in each well of a 24-well culture plate. Now, add PBS with rifampicin to the wells that do not contain vesicle-free medium to prevent bacterial growth.Place the ticks on a glass slide with double-sided carpet tape under a dissecting microscope. Before dissection, add PBS containing rifampicin to the tick and using four millimeter Vannas scissors, make an incision of approximately one millimeter on the side of each female. Now, remove the tick's dorsal side and salivary glands, then put 20 to 40 tick salivary glands in 500 microliters of vesicle-free medium added to a single well in a 24-well tissue culture treated plate.Incubate the salivary gland samples at 32 degrees Celsius for 24 hours to allow the secretion of the extracellular vesicles. At the end of the incubation, pipette all the medium containing the salivary glands into a 1.5 milliliter microcentrifuge tube and centrifuge at 300 times G at four degrees Celsius for 10 minutes. Transfer the supernatant into a new 1.5 milliliter microcentrifuge tube.Resuspend the pellet containing the salivary glands in RNA later and store at minus 80 degrees Celsius until used or indefinitely. Now, remove the cellular debris by centrifuging the supernatant at 2, 000 times G at four degrees Celsius for 10 minutes and pipette the supernatant into a new 1.5 milliliter microcentrifuge tube. Next, centrifuge at 10, 000 times G for 30 minutes at four degrees Celsius to remove apoptotic bodies and larger extracellular vesicles and transfer the supernatant to a fresh 1.5 milliliter microcentrifuge tube.Then assemble a one micrometer nylon syringe filter on a 10 milliliter syringe and keep it on an ultracentrifuge tube. Then add the sample and fill the syringe with 10 milliliters of PBS. Pass the supernatant through the filter into the tube.After filtering and balancing the tubes, place them into a 70 Ti rotor and ultracentrifuge the tubes at 100, 000 times G for 18 hours at four degrees Celsius. The concentrated extracellular vesicles form a pellet after ultracentrifugation. Remove the supernatant without disturbing the pellet and resuspend the pellet in PBS.Pipette 500 microliters of the extracellular vesicle into a 300 K centrifugal filter and centrifuge at 8, 000 times G for 10 minutes at ambient temperature and repeat this procedure until all extracellular vesicles are filtered. Finally, add 400 microliters of sterilized PBS to the column and mix thoroughly to remove extracellular vesicles attached to the membrane. Then put the sample into a 1.5 milliliter DNAase RNAse free tube.The concentration and size of extracellular vesicles varied depending on the gender and the life stage of the ticks in between the biological replicates for females, males, and nymphs. For all combined biological replicates, similar results were observed. Analysis of small RNAs isolated from the salivary gland showed bands corresponding to ribosomal RNA and microRNAs.However, the ribosomal RNAs were absent in the vesicles isolated from the salivary glands, and microRNA samples purified from extracellular vesicles showed more degradation. After enrichment, the microRNA samples purified from EVs showed minimal degradation and sufficient microRNA concentration for cDNA library synthesis. The expected band size of approximately 150 base pairs after cDNA library preparation was obtained signifies small RNA cDNA libraries.During vesicle-free preparation, ensure to follow the steps to prevent external vesicle contamination. During well plate preparation, make sure to add antibiotics to prevent bacterial contamination from the tick microbiome. The microRNAs extracted using this protocol can be used for profiling and functional experiments to validate microRNA roles such as predicting microRNA pathway targets, inhibition, and mimicry experiments.

isolation-micrornas-from-tick-ex-vivo-salivary-gland-cultures

Research

JoVE Journal

Biology

Isolation of Mouse Salivary Gland Stem Cells

Mature salivary glands of both human and mouse origin comprise a minimum of five cell types, each of which facilitates the production and excretion of saliva into the oral cavity. Serous and mucous acinar cells are the protein and mucous producing factories of the gland respectively, and represent the origin of saliva production. Once synthesised, the various enzymatic and other proteinaceous components of saliva are secreted through a series of ductal cells bearing epithelial-type morphology, until the eventual expulsion of the saliva through one major duct into the cavity of the mouth. The composition of saliva is also modified by the ductal cells during this process.
In the manifestation of diseases such as Sj&ouml;gren's syndrome, and in some clinical situations such as radiotherapy treatment for head and neck cancers, saliva production by the glands is dramatically reduced 1,2. The resulting xerostomia, a subjective feeling of dry mouth, affects not only the ability of the patient to swallow and speak, but also encourages the development of dental caries and can be socially debilitating for the sufferer.
The restoration of saliva production in the above-mentioned clinical conditions therefore represents an unmet clinical need, and as such several studies have demonstrated the regenerative capacity of the salivary glands 3-5. Further to the isolation of stem cell-like populations of cells from various tissues within the mouse and human bodies 6-8, we have shown using the described method that stem cells isolated from mouse salivary glands can be used to rescue saliva production in irradiated salivary glands 9,10. This discovery paves the way for the development of stem cell-based therapies for the treatment of xerostomic conditions in humans, and also for the exploration of the salivary gland as a microenvironment containing cells with multipotent self-renewing capabilities.

An optimized protocol for the isolation of stem cells from the mouse salivary gland is described. The method employs enzymatic and mechanical digestion, and permits isolation of salispheres containing cells with characteristics of stem cells.

Mature salivary glands of both human and mouse origin comprise a minimum of five cell types, each of which facilitates the production and excretion of ...

Vampiric Isolation of Extracellular Fluid from Caenorhabditis elegans

The genetically tractable model organism C. elegans has provided insights into a myriad of biological questions, enabled by its short generation time, ease of growth and small size. This small size, though, has disallowed a number of technical approaches found in other model systems. For example, blood transfusions in mammalian systems and grafting techniques in plants enable asking questions of circulatory system composition and signaling. The circulatory system of the worm, the pseudocoelom, has until recently been impossible to assay directly. To answer questions of intercellular signaling and circulatory system composition C. elegans researchers have traditionally turned to genetic analysis, cell/tissue specific rescue, and mosaic analysis. These techniques provide a means to infer what is happening between cells, but are not universally applicable in identification and characterization of extracellular molecules. Here we present a newly developed technique to directly assay the pseudocoelomic fluid of C. elegans. The technique begins with either genetic or physical manipulation to increase the volume of extracellular fluid. Afterward the animals are subjected to a vampiric reverse microinjection technique using a microinjection rig that allows fine balance pressure control. After isolation of extracellular fluid, the collected fluid can be assayed by transfer into other animals or by molecular means. To demonstrate the effectiveness of this technique we present a detailed approach to assay a specific example of extracellular signaling molecules, long dsRNA during a systemic RNAi response. Although characterization of systemic RNAi is a proof of principle example, we see this technique as being adaptable to answer a variety of questions of circulatory system composition and signaling.

Vampiric Isolation of Extracellular Fluid from Caenorhabditis elegans

The model organism C. elegans uses pseudocoelomic fluid as a passive circulatory system. Direct assay of this fluid has not been previously possible. Here we present a novel technique to directly assay the extracellular space, and use systemic silencing signals during an RNAi response as a proof of principle example.

The genetically tractable model organism C. elegans has provided insights into a myriad of biological questions, enabled by its short generation time, ...

Isolation of Basal Cells and Submucosal Gland Duct Cells from Mouse Trachea

The large airways are directly in contact with the environment and therefore susceptible to injury from toxins and infectious agents that we breath in 1. The large airways therefore require an efficient repair mechanism to protect our bodies. This repair process occurs from stem cells in the airways and isolating these stem cells from the airways is important for understanding the mechanisms of repair and regeneration. It is also important for understanding abnormal repair that can lead to airway diseases 2. The goal of this method is to isolate a novel stem cell population from the mouse tracheal submucosal gland ducts and to place these cells in in vitro and in vivo model systems to identify the mechanisms of repair and regeneration of the submucosal glands 3. This production shows methods that can be used to isolate and assay the duct and basal stem cells from the large airways 3.This will allow us to study diseases of the airway, such as cystic fibrosis, asthma and chronic obstructive pulmonary disease. Currently, there are no methods for isolation of submucosal gland duct cells and there are no in vivo models to study the regeneration of submucosal glands.

Here we demonstrate our protocol for isolation of basal and submucosal gland duct cells from mouse tracheas. We also demonstrate the method of injecting stem cells into the dorsal mouse fat pad to create an in vivo model of submucosal gland regeneration.

The large airways are directly in contact with the environment and therefore susceptible to injury from toxins and infectious agents that we breath in 1. ...

Genetic Modification and Recombination of Salivary Gland Organ Cultures

Branching morphogenesis occurs during the development of many organs, and the embryonic mouse submandibular gland (SMG) is a classical model for the study of branching morphogenesis. In the developing SMG, this process involves iterative steps of epithelial bud and duct formation, to ultimately give rise to a complex branched network of acini and ducts, which serve to produce and modify/transport the saliva, respectively, into the oral cavity1-3. The epithelial-associated basement membrane and aspects of the mesenchymal compartment, including the mesenchyme cells, growth factors and the extracellular matrix, produced by these cells, are critical to the branching mechanism, although how the cellular and molecular events are coordinated remains poorly understood 4. The study of the molecular mechanisms driving epithelial morphogenesis advances our understanding of developmental mechanisms and provides insight into possible regenerative medicine approaches. Such studies have been hampered due to the lack of effective methods for genetic manipulation of the salivary epithelium. Currently, adenoviral transduction represents the most effective method for targeting epithelial cells in adult glands in vivo5. However, in embryonic explants, dense mesenchyme and the basement membrane surrounding the epithelial cells impedes viral access to the epithelial cells. If the mesenchyme is removed, the epithelium can be transfected using adenoviruses, and epithelial rudiments can resume branching morphogenesis in the presence of Matrigel or laminin-1116,7. Mesenchyme-free epithelial rudiment growth also requires additional supplementation with soluble growth factors and does not fully recapitulate branching morphogenesis as it occurs in intact glands8. Here we describe a technique which facilitates adenoviral transduction of epithelial cells and culture of the transfected epithelium with associated mesenchyme. Following microdissection of the embryonic SMGs, removal of the mesenchyme, and viral infection of the epithelium with a GFP-containing adenovirus, we show that the epithelium spontaneously recombines with uninfected mesenchyme, recapitulating intact SMG glandular structure and branching morphogenesis. The genetically modified epithelial cell population can be easily monitored using standard fluorescence microscopy methods, if fluorescently-tagged adenoviral constructs are used. The tissue recombination method described here is currently the most effective and accessible method for transfection of epithelial cells with a wild-type or mutant vector within a complex 3D tissue construct that does not require generation of transgenic animals.

A technique to genetically manipulate epithelial cells within whole ex vivo cultured embryonic mouse submandibular glands (SMGs) using viral gene transfer is described. This method takes advantage of the innate ability of SMG epithelium and mesenchyme to spontaneously recombine after separation and infection of epithelial rudiments with adenoviral vectors.

Branching  morphogenesis occurs during the development of many organs, and the embryonic  mouse submandibular gland (SMG) is a classical model for the ...

Real Time Analysis of Metabolic Profile in Ex Vivo Mouse Intestinal Crypt Organoid Cultures

The small intestinal mucosa exhibits a repetitive architecture organized into two fundamental structures: villi, projecting into the intestinal lumen and composed of mature enterocytes, goblet cells and enteroendocrine cells; and crypts, residing proximal to the submucosa and the muscularis, harboring adult stem and progenitor cells and mature Paneth cells, as well as stromal and immune cells of the crypt microenvironment. Until the last few years, in vitro studies of small intestine was limited to cell lines derived from either benign or malignant tumors, and did not represent the physiology of normal intestinal epithelia and the influence of the microenvironment in which they reside. Here, we demonstrate a method adapted from Sato et al. (2009) for culturing primary mouse intestinal crypt organoids derived from C57BL/6 mice. In addition, we present the use of crypt organoid cultures to assay the crypt metabolic profile in real time by measurement of basal oxygen consumption, glycolytic rate, ATP production and respiratory capacity. Organoids maintain properties defined by their source and retain aspects of their metabolic adaptation reflected by oxygen consumption and extracellular acidification rates. Real time metabolic studies in this crypt organoid culture system are a powerful tool to study crypt organoid energy metabolism, and how it can be modulated by nutritional and pharmacological factors.

Real Time Analysis of Metabolic Profile in Ex Vivo Mouse Intestinal Crypt Organoid Cultures

Small intestinal crypt organoids cultured ex vivo provide a tissue culture system that recapitulates growth of crypts dependent on stem cells and their niche. We established a method to assay the metabolic profile in real time in primary mouse crypt organoids. We found organoids maintain physiological properties defined by their source.

The small intestinal mucosa exhibits a repetitive architecture organized into two fundamental structures: villi, projecting into the intestinal lumen and ...

Isolation of Intact, Whole Mouse Mammary Glands for Analysis of Extracellular Matrix Expression and Gland Morphology

The goal of this procedure was to harvest the #4 abdominal mammary glands from female nulliparous mice in order to assess ECM expression and ductal architecture. Here, a small pocket below the skin was created using Mayo scissors, allowing separation of the glands within the subcutaneous tissue from the underlying peritoneum. Visualization of the glands was aided by the use of 3.5x-R surgical micro loupes. The pelt was inverted and pinned back allowing identification of the intact mammary fat pads. Each of the #4 abdominal glands was bluntly dissected by sliding the scalpel blade laterally between the subcutaneous layer and the glands. Immediately post-harvest, glands were placed in 10% neutral buffered formalin for subsequent tissue processing. Excision of the entire gland is advantageous because it primarily eliminates the risk of excluding important tissue-wide interactions between ductal epithelial cells and other microenvironmental cellular populations that could be missed in a partial biopsy. One drawback of the methodology is the use of serial sections from fixed tissues which limits analyses of ductal morphogenesis and protein expression to discrete locations within the gland. As such, changes in ductal architecture and protein expression in 3 dimensions (3D) is not readily obtainable. Overall, the technique is applicable to studies requiring whole intact murine mammary glands for downstream investigations such as developmental ductal morphogenesis or breast cancer.

Here, we present a protocol for the isolation of whole, intact mouse mammary glands to investigate extracellular matrix (ECM) expression and ductal morphology. Mouse #4 abdominal glands were extracted from 8-10 week old female nulliparous mice, fixed in neutral buffered formalin, sectioned and stained using immunohistochemistry for ECM proteins.

The goal of this procedure was to harvest the #4 abdominal mammary glands from female nulliparous mice in order to assess ECM expression and ductal ...

Isolation of Retinal Arterioles for Ex Vivo Cell Physiology Studies

The retina is a highly metabolically active tissue that requires a substantial blood supply. The retinal circulation supports the inner retina, while the choroidal vessels supply the photoreceptors. Alterations in retinal perfusion contribute to numerous sight-threatening disorders, including diabetic retinopathy, glaucoma and retinal branch vein occlusions. Understanding the molecular mechanisms involved in the control of blood flow through the retina and how these are altered during ocular disease could lead to the identification of new targets for the treatment of these conditions. Retinal arterioles are the main resistance vessels of the retina, and consequently, play a key role in regulating retinal hemodynamics through changes in luminal diameter. In recent years, we have developed methods for isolating arterioles from the rat retina which are suitable for a wide range of applications including cell physiology studies. This preparation has already begun to yield new insights into how blood flow is controlled in the retina and has allowed us to identify some of the key changes that occur during ocular disease. In this article, we describe methods for the isolation of rat retinal arterioles and include protocols for their use in patch-clamp electrophysiology, calcium imaging and pressure myography studies. These vessels are also amenable for use in PCR-, western blotting- and immunohistochemistry-based studies.

Isolation of Retinal Arterioles for Ex Vivo Cell Physiology Studies

This manuscript describes a straightforward protocol for the isolation of arterioles from the rat retina that can be used in electrophysiological, calcium imaging and pressure myography studies.

The retina is a highly metabolically active tissue that requires a substantial blood supply. The retinal circulation supports the inner retina, while the ...

Flow Cytometric Analysis of Extracellular Vesicles from Cell-conditioned Media

Flow cytometry (FC) is the method of choice for semi-quantitative measurement of cell-surface antigen markers. Recently, this technique has been used for phenotypic analyses of extracellular vesicles (EV) including exosomes (Exo) in the peripheral blood and other body fluids. The small size of EV mandates the use of dedicated instruments having a detection threshold around 50-100 nm. Alternatively, EV can be bound to latex microbeads that can be detected by FC. Microbeads, conjugated with antibodies that recognize EV-associated markers/Cluster of Differentiation CD63, CD9, and CD81 can be used for EV capture. Exo isolated from CM can be analyzed with or without pre-enrichment by ultracentrifugation. This approach is suitable for EV analyses using conventional FC instruments. Our results demonstrate a linear correlation between Mean Fluorescence Intensity (MFI) values and EV concentration. Disrupting EV through sonication dramatically decreased MFI, indicating that the method does not detect membrane debris. We report an accurate and reliable method for the analysis of EV surface antigens, which can be easily implemented in any laboratory.

The protocol describes a reproducible method designed for use with cell culture supernatants to detect surface epitopes on small extracellular vesicles (EV). It utilizes specific EV immunoprecipitation using beads coupled with antibodies that recognize surface antigen CD9, CD63, and CD81. The method is optimized for downstream flow cytometry analysis.

Flow cytometry (FC) is the method of choice for semi-quantitative measurement of cell-surface antigen markers. Recently, this technique has been used for ...

Purification and Analysis of Caenorhabditis elegans Extracellular Vesicles

The secretion of small membrane-bound vesicles into the external environment is a fundamental physiological process of all cells. These extracellular vesicles (EVs) function outside the cell to regulate global physiological processes by transferring proteins, nucleic acids, metabolites, and lipids between tissues. EVs reflect the physiological state of their cells of origin. EVs are implicated to have fundamental roles in virtually every aspect of human health. Thus, EV protein and genetic cargos are being increasingly analyzed for biomarkers of health and disease. However, the EV field still lacks a tractable invertebrate model system that permits the study of EV cargo composition. C. elegans is well suited for EV research because it actively secretes EVs outside of its body into its external environment, permitting facile isolation. This article provides all the necessary information for generating, purifying, and quantifying these environmentally secreted C. elegans EVs including how to work quantitatively with very large populations of age-synchronized worms, purifying EVs, and a flow cytometry protocol that directly measures the number of intact EVs in the purified sample. Thus, the large library of genetic reagents available for C. elegans research can be tapped into for investigating the impacts of genetic pathways and physiological processes on EV cargo composition.

Purification and Analysis of Caenorhabditis elegans Extracellular Vesicles

This article presents methods for generating, purifying, and quantifying Caenorhabditis elegans extracellular vesicles.

The secretion of small membrane-bound vesicles into the external environment is a fundamental physiological process of all cells. These extracellular ...

Scalable Isolation and Purification of Extracellular Vesicles from Escherichia coli and Other Bacteria

Diverse bacterial species secrete ~20-300 nm extracellular vesicles (EVs), comprised of lipids, proteins, nucleic acids, glycans, and other molecules derived from the parental cells. EVs function as intra- and inter-species communication vectors while also contributing to the interaction between bacteria and host organisms in the context of infection and colonization. Given the multitude of functions attributed to EVs in health and disease, there is a growing interest in isolating EVs for in vitro and in vivo studies. It was hypothesized that the separation of EVs based on physical properties, namely size, would facilitate the isolation of vesicles from diverse bacterial cultures. 
The isolation workflow consists of centrifugation, filtration, ultrafiltration, and size-exclusion chromatography (SEC) for the isolation of EVs from bacterial cultures. A pump-driven tangential flow filtration (TFF) step was incorporated to enhance scalability, enabling the isolation of material from liters of starting cell culture. Escherichia coli was used as a model system expressing EV-associated nanoluciferase and non-EV-associated mCherry as reporter proteins. The nanoluciferase was targeted to the EVs by fusing its N-terminus with cytolysin A. Early chromatography fractions containing 20-100 nm EVs with associated cytolysin A - nanoLuc were distinct from the later fractions containing the free proteins. The presence of EV-associated nanoluciferase was confirmed by immunogold labeling and transmission electron microscopy. This EV isolation workflow is applicable to other human gut-associated gram-negative and gram-positive bacterial species. In conclusion, combining centrifugation, filtration, ultrafiltration/TFF, and SEC enables scalable isolation of EVs from diverse bacterial species. Employing a standardized isolation workflow will facilitate comparative studies of microbial EVs across species.

Scalable Isolation and Purification of Extracellular Vesicles from Escherichia coli and Other Bacteria

Bacteria secrete nanometer-sized extracellular vesicles (EVs) carrying bioactive biological molecules. EV research focuses on understanding their biogenesis, role in microbe-microbe and host-microbe interactions and disease, as well as their potential therapeutic applications. A workflow for scalable isolation of EVs from various bacteria is presented to facilitate standardization of EV research.

Diverse bacterial species secrete ~20-300 nm extracellular vesicles (EVs), comprised of lipids, proteins, nucleic acids, glycans, and other molecules ...