This study was supported by the National Institutes of Health, grant number R01DC012308 and R21DC018089 to HXL. We give thanks to Brett Marshall (University of Georgia, Athens, GA) and Egon Ranghini (10X GENOMICS, Pleasanton, CA) for technical assistance and consultation regarding the cell dissociation; to Francisca Gibson Burnley (University of Georgia, Athens, GA) for English editing.

Animal use (C57BL/6 mice throughout the study) was approved by the University of Georgia Institutional Animal Care and Use Committee and was in accordance with the National Institutes of Health Guidelines for care and use of animals for research.
1. Animal usage
NOTE: Mice were bred and maintained in the animal facility of the Animal and Dairy Science department at the University of Georgia at 22 &#176;C under 12-h day/night cycles.
<ol>
	<li>Designate noon of the day of vaginal plug detection in mice as embryonic (E) day 0.5. Use embryos at E12.5 and postnatal mice at 8 weeks of age for the following experiments.</li>
</ol>
2. Preparation before experiment
NOTE: The instruments required for this protocol are listed in the Table of Materials.
<ol>
	<li>Autoclave instruments before the experiment. Sterilize instruments using a bead sterilizer during the experiment.</li>
	<li>Clean the surgical area, the dissecting microscope, and the biosafety cabinet using 70% ethanol&#160;wipes. Turn on the UV light of biosafety cabinet and keep it on for 20 min prior to the experimental procedure.</li>
	<li>Prepare an enzyme mixture of 1:1 dispase (5.0 mg/mL) and collagenase (2.0 mg/mL) to a final concentration of 2.5 and 1.0 mg/mL respectively, and filter the solution using 0.22 &#181;m syringe filter7.
	<ol>
		<li>Prepare 1 mL of enzyme mixture for an adult tongue or 0.5 mL for a specific region of tongue (e.g., posterior, or anterior tongue).</li>
		<li>Prepare 2 mL of enzyme mixture for embryonic tongues.</li>
	</ol>
	</li>
	<li>Make 10 mL of 2.5% BSA in 0.1 M PBS and filter the solution using 1 mL syringe and 0.22 &#181;m syringe filter.</li>
	<li>Make 500 &#181;L of 5% FBS in DMEM/F12.</li>
	<li>Make 3 mL of DMEM/F12 containing 10% FBS and 1% BSA and filter the solution using 1 mL syringe and 0.22 &#181;m syringe filter.</li>
</ol>
3. Separation of the tongue epithelium from the mesenchyme/underlying connective tissue
<ol>
	<li>Separation of the epithelium from the mesenchyme of an E12.5 mouse tongue
	<ol>
		<li>Euthanize timed pregnant female mice carrying E12.5 embryos by placing it in a CO2 chamber followed by cervical dislocation. 
		NOTE: Mouse embryos at E12.5 are collected after 12 pm (afternoon) on the 12th day after detection of vaginal plug in the pregnant female mice.</li>
		<li>Transfer mice to the surgical area. Wet the mouse abdomen using 70% ethanol to prevent fur from getting into the operating site.</li>
		<li>Open the abdomen using dissecting scissors to expose the uterine horns carrying embryos. Dissect the uterine horns using dissecting scissors and transfer it to 15 mL of fresh Tyrode&#39;s solution in a 100 mm culture dish.</li>
		<li>Dissect the embryos (Figure 1A1) out from uterine horns under a dissecting microscope using mini-scissors and fine forceps.</li>
		<li>Carefully open the mouth cavity wide by using fine forceps and dissect the tongue off from the mandible using mini-scissors (Figure 1A2).</li>
		<li>Wash the tongues using 15 mL of fresh sterile Tyrode&#39;s solution in a 100 mm culture dish.</li>
		<li>Transfer the tissues to 2 mL of enzyme mixture of dispase (2.5 mg/mL) and collagenase (1.0 mg/mL) in a 35 mm culture dish with a spatula and fine forceps in a biosafety cabinet. Incubate for&#160;20 min at 37 &#176;C.</li>
		<li>Transfer tongues to 15 mL of fresh sterile Tyrode&#39;s solution in a 100 mm culture dish and gently remove the mesenchyme from the epithelium from the ventral side using fine forceps. 
		&#8203;NOTE: Epithelial sheets may be separated without mechanical force during the incubation.</li>
		<li>Wash the separated epithelia and mesenchyme twice in 15 mL of fresh sterile Tyrode&#39;s solution in a 100 mm culture dish. 
		NOTE: The activities of dispase and collagenase will be inhibited by EDTA in the cell dissociation procedure (step 4.1).</li>
	</ol>
	</li>
	<li>Separation of the tongue epithelium from the underlying connective tissue of adult mice
	<ol>
		<li>Euthanize the mouse at 8 weeks of age by placing it in a CO2 chamber. Confirm that the mouse is euthanized with no breaths and forepaw-pinch response.</li>
		<li>Transfer the mice to the surgical area. Wet the mouse head using 70% ethanol to prevent fur from getting into the oral cavity.</li>
		<li>Cut the corners of the mouth along the cheek using dissecting scissors to open the oral cavity. Dissect the tongue with mandible (Figure 1B1) and place it in a plastic dish with a layer of plastic wrap.</li>
		<li>Using surgical forceps to hold the tongue under a dissecting microscope, inject the enzyme mixture of dispase (2.5 mg/mL) and collagenase (1.0 mg/mL) in the sub-epithelial space of tongue through the cutting edge (Figure 1B1, arrows) of the posterior tongue.
		<ol>
			<li>Inject 1 mL of enzyme mixture evenly to the whole tongue for tissue collection from both anterior and posterior tongue.</li>
			<li>Inject 0.5 mL of enzyme mixture locally to the anterior tongue for tissue collection from tongue tip or to the posterior tongue for circumvallate papilla tissue collection. 
			&#8203;NOTE: The tongue will swell as the enzyme accumulates (Figure 1B2). Gentle injection of the enzyme can prevent pressure from damaging the epithelium and keep as much enzyme as possible in the tongue.</li>
		</ol>
		</li>
		<li>Wrap the tongue with plastic wrap and incubate the tongue for 30 min at 37 &#176;C.</li>
		<li>Use mini-scissors to dissect the tongue tip and/or circumvallate papilla, and use spatula and fine forceps to transfer tissue to 15 mL of fresh sterile Tyrode&#39;s solution in a 100 mm culture dish.</li>
		<li>Separate the epithelium from the underlying connective tissue in the enzyme-digested sub-epithelial space using mini-scissors. Trim the tissues to a proper size according to the requirement of downstream experiments.</li>
		<li>Wash the separated epithelium and underlying connective tissue twice in 15 mL of fresh sterile Tyrode&#39;s solution in a 100 mm culture dish. 
		&#8203;NOTE: The activities of dispase and collagenase will be inhibited by EDTA in the cell dissociation procedure (step 4.1).</li>
	</ol>
	</li>
</ol>
4. Cell dissociation
NOTE: The cell dissociation protocol described here can be applied to the tongue epithelium and mesenchyme/connective tissue in both E12.5 embryonic and 8-week-old mice. To reduce the cell loss during agitation and transfer of cell suspension, use commercial low retention pipette tips or pre-coated pipette tips with 2.5% BSA in 0.1 M PBS at pH 7.48.
<ol>
	<li>Transfer the tissues using spatula and fine forceps to 3 mL of 0.25% trypsin-EDTA in a new 35 mm culture dish for 30 min at 37 &#176;C. Gently agitate tissues every 5 min with 1 mL pipette tips. 
	NOTE: Do not cut the pipette tip, as the cutting edge can physically damage the dissociated cells.</li>
	<li>Add 500 &#181;L of 5% FBS in DMEM/F12 to stop the reaction and transfer the medium to a 5 mL low binding centrifuge tube.</li>
	<li>Centrifuge cell suspension at 200 x g for 8 min at room temperature and remove the supernatant.</li>
	<li>Gently re-suspend cells in 3 mL of DMEM/F12 containing 10% FBS and 1% BSA using 1 mL pipette tips and filter the cells using a 70 &#181;m cell strainer, followed by a 35 &#181;m cell strainer.</li>
	<li>Centrifuge cell suspension at 200 x g for 8 min at room temperature. Remove most of the medium and leave 50-300 &#181;L as the final volume to re-suspend cells. 
	&#8203;NOTE: Adjust the concentration of cells according to requirements of downstream experiments by changing the final volume of single cell suspension.</li>
</ol>
5. Cell counting and viability test using hemocytometer
NOTE: To improve measurement accuracy, 3 technical replicates are recommended for each sample.
<ol>
	<li>Gently mix 5-10 &#181;L of the single cell suspension with an equal volume of Trypan blue and add onto hemocytometer. 
	NOTE: Clean the hemocytometer thoroughly using 70% ethanol before use. Dusts particles on the hemocytometer will be stained in dark blue and affect the accuracy of viability test. Check the hemocytometer under a microscope.</li>
	<li>Count the total cell number, the number of live cells (white), and the number of dead cells (dark blue) stained by Trypan blue (Figure 2, arrows) respectively in 4 squares with 16 grids (Figure 2) using inverted microscope with imaging system.</li>
	<li>Calculate the cell concentration: 
	Cell concentration = <img alt="Equation 1" src="/files/ftp_upload/62163/62163eq01v2.jpg" />
	<ol>
		<li>Calculate the viability: 
		Viability = <img alt="Equation 2" src="/files/ftp_upload/62163/62163eq02.jpg" /></li>
	</ol>
	</li>
</ol>

<ol>
	<li>Grada, A., Weinbrecht, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Next-generation+sequencing:+methodology+and+application.">Next-generation sequencing: methodology and application.</a> The Journal of investigative dermatology. 133 (8), 11 (2013).</li><li>Whitley, S. K., Horne, W. T., Kolls, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Research+techniques+made+simple:+methodology+and+clinical+applications+of+RNA+sequencing.">Research techniques made simple: methodology and clinical applications of RNA sequencing.</a> Journal of Investigative Dermatology. 136 (8), 77-82 (2016).</li><li>Schaum, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+transcriptomics+of+20+mouse+organs+creates+a+Tabula+Muris:+The+Tabula+Muris+Consortium.">Single-cell transcriptomics of 20 mouse organs creates a Tabula Muris: The Tabula Muris Consortium.</a> Nature. 562 (7727), 367 (2018).</li><li>Sukumaran, S. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole+transcriptome+profiling+of+taste+bud+cells.">Whole transcriptome profiling of taste bud cells.</a> Scientific reports. 7 (1), 1-15 (2017).</li><li>Ren, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptome+analyses+of+taste+organoids+reveal+multiple+pathways+involved+in+taste+cell+generation.">Transcriptome analyses of taste organoids reveal multiple pathways involved in taste cell generation.</a> Scientific Reports. 7 (1), 1-13 (2017).</li><li>Ren, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+Lgr5-or+Lgr6-expressing+taste+stem/progenitor+cells+generate+taste+bud+cells+ex+vivo.">Single Lgr5-or Lgr6-expressing taste stem/progenitor cells generate taste bud cells ex vivo.</a> Proceedings of the National Academy of Sciences. 111 (46), 16401-16406 (2014).</li><li>Venkatesan, N., Boggs, K., Liu, H. -. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Taste+bud+labeling+in+whole+tongue+epithelial+sheet+in+adult+mice.">Taste bud labeling in whole tongue epithelial sheet in adult mice.</a> Tissue Engineering Part C: Methods. 22 (4), 332-337 (2016).</li><li>Nguyen-Ngoc, K. -. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Tissue Morphogenesis. , 135-162 (2015).</li><li>Okubo, T., Clark, C., Hogan, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+lineage+mapping+of+taste+bud+cells+and+keratinocytes+in+the+mouse+tongue+and+soft+palate.">Cell lineage mapping of taste bud cells and keratinocytes in the mouse tongue and soft palate.</a> Stem Cells. 27 (2), 442-450 (2009).</li></ol>

No conflicts of interest declared.

To date, there has not been a detailed protocol available for cell dissociation from the tongue epithelium and underlying mesenchyme/connective tissue. This current cell dissociation protocol provides a reproducible procedure to generate a single cell suspension with a high cell viability (&gt;90%) from mouse tongue tissues, including epithelial sheets and mesenchyme/connective tissues at both embryonic and postnatal stages even though isolated cells from E12.5 and adult mice are different in size. For example, isolated ...

The mammalian tongue is a complex organ critical for taste, speaking, and food processing. It is comprised of multiple types of highly organized tissues compartmentalized by mesenchyme/connective tissue and covered by a stratified epithelial sheet containing taste papillae and taste buds. Cell populations in both tongue epithelium and mesenchyme/connective tissue are heterogeneous. To better understand the functions and distribution of a particular type of cells in the tongue, studies using dissociated cells are necessary. For example, single cell RNA sequencing is a powerful and high-throughput method for transcriptomic profiling in individual cells, which is designed to understand the transcriptome of complex tissue at a single-cell resolution1,2,3,4. Primary cell culture has been proven to be a useful tool to study the function and differentiation of stem/progenitor cells for taste buds5,6. These studies require a large quantity of high-quality isolated cell populations (e.g., sufficient total cell number with proper concentration and high viability).
Thus, there is a need to isolate cells from different regions of the lingual tissues and at different developmental stages. Currently, there is not a detailed protocol available for cell dissociation from the tongue epithelium and underlying mesenchyme/connective tissue. Here, we report an optimized cell dissociation method to prepare cells for experiments requiring a high quality of live cells such as for single cell RNA sequencing and primary stem cell cultures. We found that selection of enzymatic digestion buffer, gentle pipetting, selection of resuspension medium, and optimal centrifugation time and speed are crucial to generate these large quantities of high-quality cells.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>bovine serum albumin (BSA)</td>
			<td>Gold Biotechnology</td>
			<td>A-420-100</td>
			<td></td>
		</tr>
		<tr>
			<td>C57BL/6 mouse (C57BL/6J)</td>
			<td>The Jackson Laboratory</td>
			<td>000664</td>
			<td></td>
		</tr>
		<tr>
			<td>collagenase (Collagenase A)</td>
			<td>Sigma-Aldrich</td>
			<td>10103586001</td>
			<td></td>
		</tr>
		<tr>
			<td>culture dish (35 mm in diameter)</td>
			<td>Genesee Scientific</td>
			<td>32-103G</td>
			<td></td>
		</tr>
		<tr>
			<td>culture dish (100 mm in diameter)</td>
			<td>Genesee Scientific</td>
			<td>32-107G</td>
			<td></td>
		</tr>
		<tr>
			<td>dispase (Dispase II)</td>
			<td>Sigma-Aldrich</td>
			<td>04942078001</td>
			<td></td>
		</tr>
		<tr>
			<td>dissecting scissors (Student Fine Scissors)</td>
			<td>Find Science Tool</td>
			<td>91460-11</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>Gibco</td>
			<td>11320033</td>
			<td></td>
		</tr>
		<tr>
			<td>fetal bovine serum (FBS)</td>
			<td>Hyclone</td>
			<td>C838U82</td>
			<td></td>
		</tr>
		<tr>
			<td>fine forceps (Dumount #3 Forceps)</td>
			<td>Find Science Tool</td>
			<td>11293-00</td>
			<td></td>
		</tr>
		<tr>
			<td>hemocytometer</td>
			<td>Hausser Scientific</td>
			<td>3520</td>
			<td></td>
		</tr>
		<tr>
			<td>inverted microscope with imaging system (EVOS XL Core Cell Imaging System)</td>
			<td>Life Technologies</td>
			<td>AMEX1000</td>
			<td></td>
		</tr>
		<tr>
			<td>low retention pipette tips</td>
			<td>METTLER TOLEDO</td>
			<td>17014342</td>
			<td></td>
		</tr>
		<tr>
			<td>mini-scissors (Evo Spring Scissors)</td>
			<td>Fine Science Tool</td>
			<td>15800-01</td>
			<td></td>
		</tr>
		<tr>
			<td>plastic warp</td>
			<td>VWR</td>
			<td>46610-056</td>
			<td></td>
		</tr>
		<tr>
			<td>spatula (Moria Spoon)</td>
			<td>Fine Science Tool</td>
			<td>10321-08</td>
			<td></td>
		</tr>
		<tr>
			<td>surgical forceps (Dumount #2 Laminectomy Forceps)</td>
			<td>Fine Science Tool</td>
			<td>11223-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan blue</td>
			<td>Gibco</td>
			<td>15250061</td>
			<td></td>
		</tr>
		<tr>
			<td>Tyrode&#8217;s solution</td>
			<td>Sigma-Aldrich</td>
			<td>T2145-10L</td>
			<td>made from Tyrode&#39;s salts</td>
		</tr>
		<tr>
			<td>0.25% typsin-EDTA</td>
			<td>Gibco</td>
			<td>25200056</td>
			<td></td>
		</tr>
		<tr>
			<td>0.1 M Phosphate-Buffered Saline (PBS)</td>
			<td>Hoefer</td>
			<td>33946</td>
			<td>made from 1 M PBS</td>
		</tr>
		<tr>
			<td>0.22-&#956;m syringe filter</td>
			<td>Genesee Scientific</td>
			<td>25-243</td>
			<td></td>
		</tr>
		<tr>
			<td>70% ethanol</td>
			<td>Koptec</td>
			<td>233919</td>
			<td>made from 100% ethanol</td>
		</tr>
		<tr>
			<td>1-mL syringe</td>
			<td>BD</td>
			<td>8194938</td>
			<td></td>
		</tr>
		<tr>
			<td>5-mL low binding microcentrifuge tube</td>
			<td>Eppendorf</td>
			<td>30122348</td>
			<td></td>
		</tr>
		<tr>
			<td>30-G needle</td>
			<td>BD</td>
			<td>9193532</td>
			<td></td>
		</tr>
		<tr>
			<td>35-&#956;m cell strainer</td>
			<td>Falcon</td>
			<td>64750</td>
			<td></td>
		</tr>
		<tr>
			<td>70-&#956;m cell strainer</td>
			<td>Falcon</td>
			<td>64752</td>
			<td></td>
		</tr>
	</tbody>
</table>

cell dissociation from the tongue epithelium and mesenchyme/connective tissue of embryonic-day 12.5 and 8-week-old mice

Cell dissociation has been an essential procedure for studies at the individual-cell level and/or at a cell-population level (e.g.,&#160;single cell RNA sequencing and primary cell culture). Yielding viable, healthy cells in large quantities is critical, and the optimal conditions to do so are tissue dependent. Cell populations in the tongue epithelium and underlying mesenchyme/connective tissue are heterogeneous and tissue structures vary in different regions and at different developmental stages. We have tested protocols for isolating cells from the mouse tongue epithelium and mesenchyme/connective tissue in the early developmental [embryonic day 12.5 (E12.5)] and young adult (8-week) stages. A clean separation between the epithelium and underlying mesenchyme/connective tissue was easy to accomplish. However, to further process and isolate cells, yielding viable healthy cells in large quantities, and careful selection of enzymatic digestion buffer, incubation time, and centrifugation speed and time are critical. Incubation of separated epithelium or underlying mesenchyme/connective tissue in 0.25% Trypsin-EDTA for 30 min at 37 &#176;C, followed by centrifugation at 200 x g for 8 min resulted in a high yield of cells at a high viability rate (&#62;90%) regardless of the mouse stages and tongue regions. Moreover, we found that both dissociated epithelial and mesenchymal/connective tissue cells from embryonic and adult tongues could survive in the cell culture-based medium for at least 3 h without a significant decrease of cell viability. The protocols will be useful for studies that require the preparation of isolated cells from mouse tongues at early developmental (E12.5) and young adult (8-week) stages requiring cell dissociation from different tissue compartments.

Separation of the tongue epithelium from the underlying mesenchyme/connective tissue 
In the embryonic mouse tongue, a gap in the sub-epithelial space is visible after proper enzyme digestion. Epithelial sheets of some tongues are separated without mechanical force during the incubation.
In the adult mouse tongue, a successful enzyme injection is indicated by the swelling in the injected areas (Figure 1B2), which s...

Watch this Scientific Journal Video about Cell Dissociation from the Tongue Epithelium and Mesenchyme/Connective Tissue of Embryonic-Day 12.5 and 8-Week-Old Mice at JoVE.com

Cell Dissociation from the Tongue Epithelium and Mesenchyme/Connective Tissue of Embryonic-Day 12.5 and 8-Week-Old Mice

We have developed a generalized protocol to dissociate a large quantity of high-quality single cells from the epithelium and mesenchyme/connective tissue of embryonic and adult mouse tongues.

Cell dissociation has been an essential procedure for studies at the individual-cell level and/or at a cell-population level (e.g.,&#160;single cell RNA ...

cell-dissociation-from-tongue-epithelium-mesenchymeconnective-tissue

Research

JoVE Journal

Developmental Biology

4.3K Views.  University of Georgia. We have developed a generalized protocol to dissociate a large quantity of high-quality single cells from the epithelium and mesenchyme/connective tissue of embryonic and adult mouse tongues. 

Video: Cell Dissociation from the Tongue Epithelium and Mesenchyme/Connective Tissue of Embryonic-Day 12.5 and 8-Week-Old Mice

Efficient Derivation of Retinal Pigment Epithelium Cells from Stem Cells

No cure has been discovered for age-related macular degeneration (AMD), the leading cause of vision loss in people over the age of 55. AMD is complex multifactorial disease with an unknown etiology, although it is largely thought to occur due to death or dysfunction of the retinal pigment epithelium (RPE), a monolayer of cells that underlies the retina and provides critical support for photoreceptors. RPE cell replacement strategies may hold great promise for providing therapeutic relief for a large subset of AMD patients, and RPE cells that strongly resemble primary human cells (hRPE) have been generated in multiple independent labs, including our own. In addition, the uses for iPS-RPE are not limited to cell-based therapies, but also have been used to model RPE diseases. These types of studies may not only elucidate the molecular bases of the diseases, but also serve as invaluable tools for developing and testing novel drugs. We present here an optimized protocol for directed differentiation of RPE from stem cells. Adding nicotinamide and either Activin A or IDE-1, a small molecule that mimics its effects, at specific time points, greatly enhances the yield of RPE cells. Using this technique we can derive large numbers of low passage RPE in as early as three months.

Stem cell-derived retinal pigment epithelium (RPE) cells may be used for multiple applications including cell-based therapies for retinal degeneration, disease modeling, and drug studies. Here we present a simple protocol for reproducibly deriving RPE from stem cells.

No cure has been discovered for age-related macular degeneration  (AMD), the leading cause of vision loss in people over the age of 55. AMD is complex ...

Assaying Blood Cell Populations of the Drosophila melanogaster Larva

In vertebrates, hematopoiesis is regulated by inductive microenvironments (niches). Likewise, in the invertebrate model organism&#160;Drosophila melanogaster, inductive microenvironments known as larval Hematopoietic Pockets (HPs) have been identified as anatomical sites for the development and regulation of blood cells (hemocytes), in particular of the self-renewing macrophage lineage.&#160;HPs are segmentally repeated pockets between the epidermis and muscle layers of the larva, which also comprise sensory neurons of the peripheral nervous system. In the larva, resident (sessile) hemocytes are exposed to anti-apoptotic, adhesive and proliferative cues from these sensory neurons and potentially other components of the HPs, such as the lining muscle and epithelial layers. During normal development, gradual release of resident hemocytes from the HPs fuels the population of circulating hemocytes, which culminates in the release of most of the resident hemocytes at the beginning of metamorphosis. Immune assaults, physical injury or mechanical disturbance trigger the premature release of resident hemocytes into circulation. The switch of larval hemocytes between resident locations and circulation raises the need for a common standard/procedure to selectively isolate and quantify these two populations of blood cells from single Drosophila larvae. Accordingly, this protocol describes an automated method to release and quantify the resident and circulating hemocytes from single larvae. The method facilitates ex vivo approaches, and may be adapted to serve a variety of developmental stages of Drosophila and other invertebrate organisms.

Assaying Blood Cell Populations of the Drosophila melanogaster Larva

Drosophila blood cells, or hemocytes, cycle between resident sites and circulation. In the larva, resident (sessile) hemocytes localize to inductive microenvironments, the Hematopoietic Pockets, while circulating hemocytes move freely in the hemolymph. The goal of this protocol is the standardized isolation and quantification of these two, behaviorally distinct but interchanging, hemocyte populations.

In vertebrates, hematopoiesis is regulated by inductive microenvironments (niches). Likewise, in the invertebrate model organism&#160;Drosophila ...

Isolation of Perivascular Multipotent Precursor Cell Populations from Human Cardiac Tissue

Multipotent mesenchymal stem/stromal cells (MSC) were conventionally isolated, through their plastic adherence, from primary tissue digests whilst their anatomical tissue location remained unclear. The recent discovery of defined perivascular and MSC cell marker expression by perivascular cells in multiple tissues by our group and other researchers has provided an opportunity to prospectively isolate and purify specific homogenous subpopulations of multipotent perivascular precursor cells. We have previously demonstrated the use of fluorescent activated cell sorting (FACS) to purify microvascular CD146+CD34- pericytes and vascular CD34+CD146- adventitial cells from human skeletal muscle. Herein we describe a method to simultaneously isolate these two perivascular cell subsets from human myocardium by FACS, based on the expression of a defined set of cell surface markers for positive and negative selections. This method thus makes available two specific subpopulations of multipotent cardiac MSC-like precursor cells for use in basic research and/or therapeutic investigations.

Human cardiac tissue harbours multipotent perivascular precursor cell populations that may be suitable for myocardial regeneration. The technique described here allows for the simultaneous isolation and purification of two multipotent stromal cell populations associated with native blood vessels, i.e. CD146+CD34- pericytes and CD34+CD146- adventitial cells, from the human myocardium.

Multipotent mesenchymal stem/stromal cells (MSC) were conventionally isolated, through their plastic adherence, from primary tissue digests whilst their ...

A Simplified and Efficient Method to Isolate Primary Human Keratinocytes from Adult Skin Tissue

Primary human keratinocytes isolated from fresh skin tissues and their expansion in vitro have been widely used for laboratory research and for clinical applications. The conventional isolation method of human keratinocytes involves a two-step sequential enzymatic digestion procedure, which has been proven to be inefficient in generating primary cells from adult tissues due to the low cell recovery rate and reduced cell viability. We recently reported an advanced method to isolate human primary epidermal progenitor cells from skin tissues that utilizes the Rho kinase inhibitor Y-27632 in the medium. Compared with the traditional protocol, this new method is simpler, easier, and less time-consuming, and increases epithelial stem cell yield and enhances their stem cell characteristics. Moreover, the new methodology does not require the separation of the epidermis from the dermis, and, therefore, is suitable for isolating cells from different types of adult tissues. This new isolation method overcomes the major shortcomings of conventional methods and is more suitable for producing large numbers of epidermal cells with high potency both for laboratory and for clinical applications. Here, we describe the new method in detail.

Here we present a protocol to efficiently isolate primary human keratinocytes from adult skin tissues. This method simplifies the conventional procedure by using the ROCK Inhibitor Y-27632 in the inoculation medium to spontaneously separate epidermal cells from dermal cells.

Primary human keratinocytes isolated from fresh skin tissues and their expansion in vitro have been widely used for laboratory research and for clinical ...

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

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

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

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

Murine Neural Plate Targeting by In Utero Nano-Injection (NEPTUNE) at Embryonic Day 7.5

Manipulating gene expression in the developing mouse brain in utero holds great potential for functional genetics studies. However, it has previously largely been restricted to the manipulation of embryonic stages post-neurulation. A protocol was developed to inject the amniotic cavity at embryonic day (E)7.5 and deliver lentivirus, encoding cDNA or shRNA, targeting &gt;95% of the neural plate and neural crest cells, contributing to the future brain, spinal cord, and peripheral nervous system. This protocol describes the steps necessary to achieve successful transduction, including grinding of the glass capillary needles, pregnancy verification, developmental staging using ultrasound imaging, and optimal injection volumes matched to embryonic stages. 
Following this protocol, it is possible to achieve transduction of &gt;95% of the developing brain with high-titer lentivirus and thus perform whole-brain genetic manipulation. In contrast, it is possible to achieve mosaic transduction using lower viral titers, allowing for genetic screening or lineage tracing. Injection at E7.5 also targets ectoderm and neural crest contributing to distinct compartments of the eye, tongue, and peripheral nervous system. This technique thus offers the possibility to manipulate gene expression in mouse neural-plate- and ectoderm-derived tissues from preneurulation stages, with the benefit of reducing the number of mice used in experiments.

Murine Neural Plate Targeting by In Utero Nano-Injection NEPTUNE at Embryonic Day 7.5

In this protocol, we describe how to inject the mouse amniotic cavity at E7.5 with lentivirus, leading to uniform transduction of the entire neural plate, with minimal detrimental effects on survival or embryonic development.

Manipulating gene expression in the developing mouse brain in utero holds great potential for functional genetics studies. However, it has previously ...

Microdissection and Dissociation of the Murine Oviduct: Individual Segment Identification and Single Cell Isolation

Mouse model systems are unmatched for the analysis of disease processes because of their genetic manipulability and the low cost of experimental treatments. However, because of their small body size, some structures, such as the oviduct with a diameter of 200-400 &#956;m, have proven to be relatively difficult to study except by immunohistochemistry. Recently, immunohistochemical studies have uncovered more complex differences in oviduct segments than were previously recognized; thus, the oviduct is divided into four functional segments with different ratios of seven distinct epithelial cell types. The different embryological origins and ratios of the epithelial cell types likely make the four functional regions differentially susceptible to disease. For example, precursor lesions to serous intraepithelial carcinomas arise from the infundibulum in mouse models and from the corresponding fimbrial region in the human fallopian tube. The protocol described here details a method for microdissection to subdivide the oviduct in such a way to yield a sufficient amount and purity of RNA necessary for downstream analysis such as reverse transcription-quantitative PCR (RT-qPCR) and RNA sequencing (RNAseq). Also described is a mostly non-enzymatic tissue dissociation method appropriate for flow cytometry or single cell RNAseq analysis of fully differentiated oviductal cells. The methods described will facilitate further research utilizing the murine oviduct in the field of reproduction, fertility, cancer, and immunology.

A method for microdissection of the mouse oviduct that allows collection of the individual segments while maintaining RNA integrity is presented. In addition, non-enzymatic oviductal cell dissociation procedure is described. The methods are appropriate for subsequent gene and protein analysis of the functionally different oviductal segments and dissociated oviductal cells.

Mouse model systems are unmatched for the analysis of disease processes because of their genetic manipulability and the low cost of experimental ...

Development of Organoids from Mouse Pituitary as In Vitro Model to Explore Pituitary Stem Cell Biology

The pituitary is the master endocrine gland regulating key physiological processes, including body growth, metabolism, sexual maturation, reproduction, and stress response. More than a decade ago, stem cells were identified in the pituitary gland. However, despite the application of transgenic in vivo approaches, their phenotype, biology, and role remain unclear. To tackle this enigma, a new and innovative organoid in vitro model is developed to deeply unravel pituitary stem cell biology. Organoids represent 3D cell structures that, under defined culture conditions, self-develop from a tissue's (epithelial) stem cells and recapitulate multiple hallmarks of those stem cells and their tissue. It is shown here that mouse pituitary-derived organoids develop from the gland's stem cells and faithfully recapitulate their in vivo phenotypic and functional characteristics. Among others, they reproduce the activation state of the stem cells as in vivo occurring in response to transgenically inflicted local damage. The organoids are long-term expandable while robustly retaining their stemness phenotype. The new research model is highly valuable to decipher the stem cells' phenotype and behavior during key conditions of pituitary remodeling, ranging from neonatal maturation to aging-associated fading, and from healthy to diseased glands. Here, a detailed protocol is presented to establish mouse pituitary-derived organoids, which provide a powerful tool to dive into the yet enigmatic world of pituitary stem cells.

Development of Organoids from Mouse Pituitary as In Vitro Model to Explore Pituitary Stem Cell Biology

The pituitary gland is the key regulator of the body's endocrine system. This article describes the development of organoids from the mouse pituitary as a novel 3D in vitro model to study the gland's stem cell population of which the biology and function remain poorly understood.

The pituitary is the master endocrine gland regulating key physiological processes, including body growth, metabolism, sexual maturation, reproduction, ...

Establishing Organoids from Human Tooth as a Powerful Tool Toward Mechanistic Research and Regenerative Therapy

Teeth are of key importance in life not only for food mastication and speech but also for psychological well-being. Knowledge on human tooth development and biology is scarce. In particular, not much is known about the tooth's epithelial stem cells and their function. We succeeded in developing a novel organoid model starting from human tooth tissue (i.e., dental follicle, isolated from extracted wisdom teeth). The organoids are robustly and long-term expandable and recapitulate the proposed human tooth epithelial stem cell compartment in terms of marker expression as well as functional activity. In particular, the organoids are capable to unfold an ameloblast differentiation process as occurring in vivo during amelogenesis. This unique organoid model will provide a powerful tool to study not only human tooth development but also dental pathology, and may pave the way toward tooth-regenerative therapy. Replacing lost teeth with a biological tooth based on this new organoid model could be an appealing alternative to the current standard implantation of synthetic materials.

We present a protocol to develop epithelial organoid cultures starting from human tooth. The organoids are robustly expandable and recapitulate the tooth's epithelial stem cells, including their ameloblast differentiation capacity. The unique organoid model provides a promising tool to study human dental (stem cell) biology with perspectives for tooth-regenerative approaches.

Teeth are of key importance in life not only for food mastication and speech but also for psychological well-being. Knowledge on human tooth development ...

Identification and Isolation of Burst-Forming Unit and Colony-Forming Unit Erythroid Progenitors from Mouse Tissue by Flow Cytometry

Early erythroid progenitors were originally defined by their colony-forming potential in vitro and classified into burst-forming and colony-forming &#34;units&#34; known as BFU-e and CFU-e. Until recently, methods for the direct prospective and complete isolation of pure BFU-e and CFU-e progenitors from freshly isolated adult mouse bone marrow were not available. To address this gap, a single-cell RNA-seq (scRNAseq) dataset of mouse bone marrow was analyzed for the expression of genes coding for cell surface markers. This analysis was combined with cell fate assays, allowing the development of a novel flow cytometric approach that identifies and allows the isolation of complete and pure subsets of BFU-e and CFU-e progenitors in mouse bone marrow or spleen. This approach also identifies other progenitor subsets, including subsets enriched for basophil/mast cell and megakaryocytic potentials. The method consists of labeling fresh bone marrow or spleen cells with antibodies directed at Kit and CD55. Progenitors that express both these markers are then subdivided into five principal populations. Population 1 (P1 or CFU-e, Kit+ CD55+ CD49fmed/low CD105med/high CD71med/high) contains all of the CFU-e progenitors and may be further subdivided into P1-low (CD71med CD150high) and P1-hi (CD71high CD150low), corresponding to early and late CFU-e, respectively; Population 2 (P2 or BFU-e, Kit+ CD55+ CD49fmed/low CD105med/high CD71low CD150high) contains all of the BFU-e progenitors; Population P3 (P3, Kit+ CD55+ CD49fmed/high CD105med/low CD150low CD41low) is enriched for basophil/mast cell progenitors; Population 4 (P4, Kit+ CD55+ CD49fmed/high CD105med/low CD150high CD41+) is enriched for megakaryocytic progenitors; and Population 5 (P5, Kit+ CD55+ CD49fmed/high CD105med/low CD150high CD41-) contains progenitors with erythroid, basophil/mast cell, and megakaryocytic potential (EBMP) and erythroid/ megakaryocytic/ basophil-biased multipotential progenitors (MPPs). This novel approach allows greater precision when analyzing erythroid and other hematopoietic progenitors and also allows for reference to transcriptome information for each flow cytometrically defined population.

Here, we describe a novel flow cytometric method for prospective isolation of early burst-forming unit erythroid (BFU-e) and colony-forming unit erythroid (CFU-e) progenitors directly from fresh mouse bone marrow and spleen. This protocol, developed based on single-cell transcriptomic data, is the first to isolate all the tissue's erythroid progenitors with high purity.

Early erythroid progenitors were originally defined by their colony-forming potential in vitro and classified into burst-forming and colony-forming ...