This work was supported by grants from the KU Leuven Research Fund and the Fund for Scientific Research (FWO) - Flanders. E.L. (11A3320N), and C.N. (1S14218N) are supported by a Ph.D. Fellowship from the FWO/FWO-SB.

Animal experiments for this study were approved by the KU Leuven Ethical Committee for Animal Experimentation (P153/2018). All mice were housed at the university&#39;s animal facility under standardized conditions (constant temperature of 23 &#177; 1.5 &#176;C, relative humidity 40%-60%, and a day/night cycle of 12 h), with access to water and food ad libitum.
1. Mice
<ol>
	<li>Use commercially available mouse strains, such as C57BL/6J mice, of young-adult age (8-12 weeks old). In general, 2-3 mice provide a sufficient number of AL cells for the protocol.</li>
</ol>
2. Isolation and dissociation of mouse AL
NOTE: Medium A, B, and C are prepared in advance19,20. Compositions are shown in Table 2.
<ol>
	<li>Isolation of mouse AL
	<ol>
		<li>Euthanize the mice by CO2 asphyxiation, followed by decapitation (Figure 2A). Wash mice heads with deionized water to remove the blood and spray them with 70% EtOH to generate a sterile environment.</li>
		<li>Using sterile surgical tools, remove the skin of the head between the ears (Figure 2B).</li>
		<li>Open the cranium and remove the brain.
		<ol>
			<li>Break the &#39;nose bridge&#39; (i.e., anterior part of the frontal bone; Figure 2B) with sterile scissors.</li>
			<li>Open the cranium further with scissors, starting from the broken nose bridge toward the ears, on both sides (Figure 2C).</li>
			<li>Remove the cranium and the brain with sterile tweezers, without touching the pituitary gland (Figure 2D).</li>
		</ol>
		</li>
		<li>Remove the diaphragma sellae with blunt tweezers, without damaging the pituitary. Discard the PL and the IL from the AL under a stereomicroscope. 
		NOTE: The PL and IL are linked and thus removed simultaneously. These parts appear as white tissue, as compared to the pink-colored AL (Figure 2D).</li>
		<li>Carefully isolate the AL with blunt tweezers and collect it in a 10 mL Erlenmeyer flask, filled with 3 mL of medium A (see Table 2). Place the flask on ice until further processing.</li>
	</ol>
	</li>
	<li>Dissociation of mouse AL
	<ol>
		<li>Remove the supernatant (SN) medium A from the Erlenmeyer flask containing the isolated AL. Add 2 mL of prewarmed (37 &#176;C) 2.5% trypsin solution and incubate at 37 &#176;C for 15 min.</li>
		<li>Without removing the trypsin solution, add 2 mL of prewarmed (37 &#176;C) DNase solution (2 &#181;g/mL in medium A; sterile-filtered through a 0.22 &#181;m mesh), and swirl the Erlenmeyer flask 10 times. Let the pituitary sink to the bottom (~1 min) and remove the SN.</li>
		<li>Add 2 mL of prewarmed (37 &#176;C) trypsin inhibitor solution (0.1 mg/mL in medium A; sterile-filtered through a 0.22 &#181;m mesh) and incubate at 37 &#176;C for 10 min. Let the pituitary sediment to the bottom and remove the SN.</li>
		<li>Add 2 mL of prewarmed (37 &#176;C) medium B (see Table 2) and incubate at 37 &#176;C for 5 min. Without removing the SN, add 2 mL of prewarmed (37 &#176;C) medium C (see Table 2) and incubate at 37 &#176;C for 15 min.</li>
		<li>Let the pituitary sink to the bottom and remove the SN. Rinse the pituitary three times with prewarmed (37 &#176;C) medium C.</li>
		<li>Dissociate the pituitary into single cells.
		<ol>
			<li>Add 2 mL of prewarmed (37 &#176;C) medium C. Aspirate and expel the pituitary gland with a sterile, flame-polished Pasteur pipette multiple times, until fragments are not visible anymore.</li>
			<li>Transfer the suspension to a 15 mL tube with 4.5 mL of prewarmed (37 &#176;C) DNase solution (2 &#181;g/mL in medium A; sterile-filtered through a 0.22 &#181;m mesh). Rinse the Erlenmeyer three times with 2 mL of prewarmed (37 &#176;C) medium C and transfer the suspension to the 15 mL tube.</li>
		</ol>
		</li>
		<li>Mix the collected cell suspension and filter it through a 40 &#181;m cell strainer into a 30 mL tube. Rinse the 15 mL tube and the cell strainer three times with 2 mL of medium C and transfer the suspension to the 30 mL tube.</li>
		<li>Position the tip of a glass Pasteur pipette, filled with 2 mL of 3% bovine serum albumin (BSA) solution (in medium A; sterile-filtered through a 0.22 &#181;m mesh), at the bottom of the tube and gently pipette out to form a visible density layer. Centrifuge at 190 x g for 10 min at 4 &#176;C.</li>
		<li>Remove the SN by inverting the tube in one fluent movement and remove the remaining SN droplets with a P1000 tip. Resuspend the cell pellet in 1 mL of ice-cold Advanced DMEM/F12 (Adv DMEM/F12) and quantify the cells with a cell counter.</li>
	</ol>
	</li>
</ol>
3. Establishment and culturing of AL-derived organoids
NOTE: Thaw ECM on ice in advance (2-3 h for 1 mL) and keep it on ice for the duration of the protocol.
<ol>
	<li>Organoid seeding and culturing
	<ol>
		<li>Centrifuge the AL cell suspension at 190 x g for 10 min at 4 &#176;C and remove the SN. Resuspend the cell pellet in Adv DMEM/F12&#160;using the specific volume calculated to reach a cell density of 1.1 x 106 cells/mL. 
		&#8203;NOTE: For instance, if the cell suspension contains 500,000 cells/mL, one must resuspend the cell pellet in 454.54 &#181;L of Adv DMEM/F12&#160;to reach the desired density of 1.1 x 106 cells/mL.</li>
		<li>Take out the volume of cell suspension needed for plating (according to the desired number of wells to seed for organoid development) and add ECM in a 30:70 ratio (30% cell suspension (in Adv DMEM/F12&#160;) and 70% ECM). Mix well by pipetting up and down. 
		NOTE: For instance, for one droplet of 30 &#181;L (see step 3.1.3), one should (gently) mix 9 &#181;L of cell suspension (containing ~10,000 cells when taken from the 1.1 x 106 cells/mL suspension) with 21 &#181;L of ECM.</li>
		<li>Per well, deposit a 30 &#181;L drop of the cell suspension/ECM mixture (see step 3.1.2) on a pre-warmed (37 &#176;C) 48-well plate. Turn the plate upside down and let the ECM solidify at 37 &#176;C for 20 min. 
		NOTE: Pre-warm the culture plates for at least 24 h at 37 &#176;C.</li>
		<li>Return the plate to its proper orientation and carefully add 250 &#181;L of prewarmed (37 &#176;C) PitOM (see Table 1) supplemented with 10 &#181;M Rock Inhibitor (Y-27632).</li>
		<li>Continue to culture the organoids by changing the medium (devoid of Y-27632) every 2-3 days until the organoids are fully grown, which takes between 10-14 days (Figure 3A). Then, passage the organoids. 
		NOTE: When aspirating the medium, make sure not to disrupt the ECM dome. Tilt the culture plate slightly and remove the medium from the bottom rim of the well. Fresh (prewarmed at 37 &#176;C) medium should be added gently to the side of the well. If gel droplets de-attach, collect the organoids and resuspend and culture them again in a new ECM droplet.</li>
	</ol>
	</li>
	<li>Organoid passaging
	<ol>
		<li>Aspirate the medium gently and add 400 &#181;L of ice-cold Adv DMEM/F12&#160;to disintegrate the ECM and collect the organoids in a microcentrifuge tube. Wash once with 400 &#181;L of ice-cold Adv DMEM/F12&#160;EM. Centrifuge at 200 x g for 5 min at 4 &#176;C.</li>
		<li>Remove the SN carefully and add 400 &#181;L of prewarmed (37 &#176;C) TrypLE Express Enzyme (1X). Mix by inverting the tube several times, and incubate at 37 &#176;C for 5 min.</li>
		<li>Add 400 &#181;L of ice-cold Adv DMEM/F12&#160;and centrifuge at 200 x g for 5 min at 4 &#176;C. Remove the SN.</li>
		<li>Resuspend the pellet with 100 &#181;L of ice-cold Adv DMEM/F12&#160;and subsequently break up the organoids by vigorously pipetting up and down with a narrowed P200 tip (i.e., push down the empty tip against the bottom of the microcentrifuge tube, to reduce its opening diameter) until organoid fragments (with a diameter around 50 &#181;m) are obtained (Figure 3B). 
		NOTE: The dissociation mixture should contain predominantly organoid fragments and only a few single cells. Harsh dissociation of the organoids into single cells negatively impacts the re-growth of the organoids.</li>
		<li>Add 800 &#181;L of Adv DMEM/F12&#160;and centrifuge at 190 x g for 10 min at 4 &#176;C. Remove the SN.</li>
		<li>Passage the organoids in a 1:2 to 1:4 ratio. Resuspend the pellet in an adequate volume of Adv DMEM/F12&#160;as needed for plating and add ECM in a 30:70 ratio (30% cell suspension and 70% ECM). Mix well by pipetting up and down.</li>
		<li>Seed and culture the organoids as described above in steps 3.1.3-3.1.5. 
		&#8203;NOTE: On average, 20 organoids develop per well from the 10,000 whole-AL cells seeded (0.2%). These passage 0 organoids can be split in a 1:2 ratio, resulting in &#62;50 organoids developing per well (passage 1). Organoids can then be split in a 1:2 to 1:4 ratio during subsequent passages. Re-growth of the organoids slows down after ~10 passages (corresponding to 3 months of culture), concretized in gradually fewer and smaller organoids.</li>
	</ol>
	</li>
</ol>
4. Cryopreservation of AL-derived organoids and thawing
<ol>
	<li>Cryopreservation of organoids
	<ol>
		<li>Follow the passaging protocol from step 3.2.1 until step 3.2.5.</li>
		<li>Resuspend the organoid pellet (containing fragments and cells) with 1 mL of cryopreservation medium (Table 3). Transfer the suspension into a cryovial and place it on ice. 
		&#8203;NOTE: Organoids (i.e., resultant fragments and cells) from up to four wells of the 48-well plate can be combined in one cryovial.</li>
		<li>Place the cryovials in a freezing container and transfer them to -80 &#176;C.</li>
		<li>After 24 h, transfer the samples to a cryobox and store them in liquid nitrogen (-196 &#176;C) for long-term storage.</li>
	</ol>
	</li>
	<li>Thawing of cryopreserved organoids
	<ol>
		<li>Remove the cryovial from the liquid nitrogen tank and place it on ice. Immediately proceed with the thawing protocol.</li>
		<li>Thaw the solution with the cryopreserved organoid fragments and single cells at 37 &#176;C (water bath). 
		NOTE: Do not keep the solution for more than 2 min at 37 &#176;C to avoid cell toxicity by DMSO.</li>
		<li>Transfer the content to a 15 mL tube containing 10 mL of ice-cold Adv DMEM/F12&#160;with 30% fetal bovine serum (FBS). Rinse the cryovial with 1 mL of Adv DMEM/F12&#160;with 30% FBS.</li>
		<li>Centrifuge at 190 x g for 10 min at 4 &#176;C. Resuspend the pellet with 1 mL of ice-cold Adv DMEM/F12&#160;and transfer the suspension to a microcentrifuge tube.</li>
		<li>Centrifuge at 190 x g for 10 min at 4 &#176;C. Resuspend the pellet in an adequate volume of Adv DMEM/F12&#160;as needed for plating and add ECM in a 30:70 ratio. Mix well by pipetting up and down.</li>
		<li>Seed and culture the organoids as described above in steps 3.1.3-3.1.5.</li>
	</ol>
	</li>
</ol>
5. Validation of AL-derived organoids
<ol>
	<li>Collection and lysis of organoids for RNA isolation
	<ol>
		<li>Collect and centrifuge the organoids as described above (step 3.2.1).</li>
		<li>Remove the SN and add 350 &#181;L of lysis buffer with 1% 2-mercapto-ethanol. Vortex for 30 s and store at -80 &#176;C or proceed immediately to RNA isolation. 
		&#8203;CAUTION: Beware that 2-mercapto-ethanol is a toxic compound. All work must be done in a chemical fume hood while wearing nitrile gloves, a dust mask, and safety glasses. 2-Mercapto-ethanol can cause irreversible damage to the eyes and skin.</li>
	</ol>
	</li>
	<li>Fixation and embedding of organoids for immuno-histochemistry/-fluorescence staining
	<ol>
		<li>Collect and centrifuge the organoids as described above (step 3.2.1).</li>
		<li>Remove the SN, add 1 mL of 4% paraformaldehyde (PFA) and incubate for 30 min at room temperature (RT) on an orbital shaker (100 rpm). 
		CAUTION: PFA is a known human carcinogen that can cause irreversible damage to the cornea. All work must be done in a chemical fume hood. Nitrile gloves and safety glasses must always be worn.</li>
		<li>Centrifuge at 200 x g for 5 min and remove the SN. Add 1 mL of PBS, incubate 10 min at RT on an orbital shaker (100 rpm), and centrifuge at 90 x g for 3 min&#160;at 4 &#176;C. Repeat the washing step twice. Store in PBS at 4 &#176;C.</li>
		<li>For tissue processing and dehydration, remove the SN and add 150 &#181;L of 2% agarose gel (in PBS) to the organoid pellet using a prewarmed widened p200 tip (made by cutting a small piece of the tip). Immediately pipet the entire volume up and eject in the lid of the microcentrifuge tube. 
		NOTE: It is important to work swiftly, as the gel containing the organoids will quickly solidify.</li>
		<li>Let the gel firmly solidify for 30 min and move the gel disc to a histology cassette. Immerse and store in 50% EtOH, until dehydration in the tissue processor.</li>
		<li>For paraffin embedding, place the gel disc (using forceps) in an embedding mold and fill with warm paraffin (60 &#176;C). Place the molds at 4 &#176;C until the paraffin is solid (approximately 45 min). These samples can either be stored at 4 &#176;C or can immediately be subjected to sectioning.</li>
		<li>Microtome the paraffin blocks containing organoids at 5 &#181;m thickness and collect the samples on glass slides. Add one drop of deionized water underneath each section to allow proper stretching of the section and place the slides on a flat heating plate at 37 &#176;C overnight. Store the slides with sections at 4 &#176;C or directly continue with immunohistochemical or immunofluorescence staining.</li>
	</ol>
	</li>
</ol>

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L., Levy, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anterior+pituitary+cell+population+control:+basal+cell+turnover+and+the+effects+of+adrenalectomy+and+dexamethasone+treatment.">Anterior pituitary cell population control: basal cell turnover and the effects of adrenalectomy and dexamethasone treatment.</a> Journal of Neuroendocrinology. 10 (3), 207-215 (1998).</li></ol>

The authors declare no competing financial interests.

The AL-derived organoids, as described here, represent a powerful research model to study pituitary stem cells in vitro. At present, this organoid approach is the only available tool to reliably and robustly grow and expand primary pituitary stem cells. A pituitary organoid model derived from embryonic stem cells (ESC) or induced pluripotent stem cells (iPSC) has been reported previously, which closely recapitulates pituitary embryonic organogenesis23; however, although highly useful to s...

The pituitary is a tiny endocrine gland located at the base of the brain, where it is connected to the hypothalamus. The gland integrates peripheral and central (hypothalamic) inputs to generate a tuned and coordinated hormone release, thereby regulating downstream target endocrine organs (such as adrenal glands and gonads) for producing appropriate hormones at the proper time. The pituitary is the key regulator of the endocrine system and is therefore rightfully termed the master gland1.
The mouse pituitary consists of three lobes (Figure 1), i.e., the anterior lobe (AL), the intermediate lobe (IL), and the posterior lobe (PL). The major endocrine AL contains five hormonal cell types, including somatotropes that produce growth hormone (GH); lactotropes generating prolactin (PRL); corticotropes that secrete adrenocorticotropic hormone (ACTH); thyrotropes responsible for thyroid-stimulating hormone (TSH) production; and gonadotropes that make luteinizing hormone (LH) and follicle-stimulating hormone (FSH). The PL consists of axonal projections from the hypothalamus in which the hormones oxytocin and vasopressin (antidiuretic hormone) are stored. The IL is located in-between the AL and PL and houses melanotropes that produce melanocyte-stimulating hormone (MSH). In the human pituitary, the IL regresses during development, and melanotropes are spread within the AL1. In addition to the endocrine cells, the pituitary gland also contains a pool of stem cells, essentially marked by the transcription factor SOX22,3,4,5,6. These SOX2+ cells are located in the marginal zone (MZ), the epithelial lining of the cleft (an embryonic remnant lumen between the AL and IL), or are spread as clusters throughout the parenchyma of the AL, thereby proposing two stem cell niches in the gland (Figure 1)2,3,4,5,6.
Given the indispensable nature of the pituitary, malfunctioning of the gland is associated with serious morbidity. Hyperpituitarism (characterized by over-secretion of one or more hormones) and hypopituitarism (defective or missing production of one or more hormones) can be caused by pituitary neuroendocrine tumors (PitNETs; e.g., ACTH-producing tumors leading to Cushing&#39;s disease) or by genetic defects (e.g., GH deficiency resulting in dwarfism)7. In addition, pituitary surgery (e.g., to remove tumors), infections (e.g., hypothalamic-pituitary tuberculosis, or infections following bacterial meningitis or encephalitis), Sheehan&#39;s syndrome (necrosis because of insufficient blood flow due to heavy blood loss at birth-giving), pituitary apoplexy and traumatic brain injury are other important causes of pituitary hypofunction8. It has been shown that the mouse pituitary possesses the regenerative capacity, being able to repair local damage introduced by transgenic ablation of endocrine cells9,10. The SOX2+ stem cells acutely react to the inflicted injury showing an activated phenotype, marked by enhanced proliferation (resulting in stem cell expansion) and increased expression of stemness-related factors and pathways (e.g., WNT/NOTCH). Moreover, the stem cells start to express the ablated hormone, finally resulting in substantial restoration of the depleted cell population over the following (5 to 6) months9,10. Also, during the neonatal maturation phase of the gland (the first 3 weeks after birth), the pituitary stem cells are thriving in an activated state6,11,12,13, whereas organismal aging is associated with declined in situ stem cell functionality, due to an increasing inflammatory (micro-) environment at aging (or &#39;inflammaging&#39;)10,14. In addition, tumorigenesis in the gland is also associated with stem cell activation7,15. Although stem cell activation has been detected in several situations of pituitary remodeling (reviewed in7,16), underlying mechanisms remain unclear. Since in vivo approaches (such as lineage tracing in transgenic mice) have not delivered a clear or comprehensive picture of pituitary stem cells, the development of reliable in vitro models to explore stem cell biology in normal and diseased pituitary is essential. Standard in vitro culture of primary pituitary stem cells remains inadequate because of very limited growth capacity and non-physiological (2D) conditions with rapid loss of phenotype (for a more detailed overview, see16). 3D sphere cultures (pituispheres) have been established from pituitary stem cells as identified by side population and SOX2+ phenotype2,3,4. The pituispheres clonally grow from the stem cells, express stemness markers and show differentiation capacity into the endocrine cell types. However, they do not considerably expand while showing only limited passageability (2-3 passages)3,4. Sphere-like structures were also obtained from non-dissociated pituitary stem cell clusters when cultured in 50% diluted Matrigel for 1 week, but expandability was not shown17. The pituisphere approach is mostly used as a read-out tool for stem cell numbers, but further applications are limited by inferior expansion capacity16.
To address and overcome these shortcomings, a new 3D model has recently been established, i.e., organoids, starting from the major endocrine AL of mice containing the MZ and parenchymal stem cells. It has been shown that the organoids are indeed derived from the pituitary&#39;s stem cells and faithfully recapitulate their phenotype18. Moreover, the organoids are long-term expandable, while robustly maintaining their stemness nature. Therefore, they provide a reliable method to expand primary pituitary stem cells for profound exploration. Such exploration is not achievable with the limited number of stem cells that can be isolated from a pituitary, which are also not expandable in 2D conditions16. It has been shown that the organoids are valuable and reliable tools to uncover new pituitary stem cell features (translatable to in vivo)14,18. Importantly, the organoid model faithfully mirrors the pituitary stem cell activation status as occurring during local tissue damage and neonatal maturation, showing enhanced formation efficiency and replicating upregulated molecular pathways14,18. Hence, the pituitary-derived organoid model is an innovative and powerful pituitary stem cell biology research model as well as a stem cell activation readout tool.
This protocol describes in detail the establishment of mouse pituitary-derived organoids. To this aim, the AL is isolated and dissociated into single cells, which are embedded in extracellular matrix-mimicking Matrigel (hereon referred to as ECM). The cell-ECM assembly is then cultured in a defined medium, essentially containing stem cell growth factors and pituitary embryonic regulators (further referred to as &#39;pituitary organoid medium&#39; (PitOM)18; Table 1). Once the organoids are fully developed (after 10-14 days), they can be further expanded trough sequential passaging and subjected to extensive downstream exploration (e.g., immunofluorescence, RT-qPCR, and bulk or single-cell transcriptomics; Figure 1). In the longer run, it is expected that the pituitary stem cell organoids will pave the way to tissue repair approaches and regenerative medicine.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Sigma-Aldrich</td>
			<td>M6250</td>
			<td></td>
		</tr>
		<tr>
			<td>48-well plates, TC treated, individually wrapped</td>
			<td>Costar</td>
			<td>734-1607</td>
			<td></td>
		</tr>
		<tr>
			<td>A83-01</td>
			<td>Sigma-Aldrich</td>
			<td>SML0788</td>
			<td></td>
		</tr>
		<tr>
			<td>Advanced DMEM</td>
			<td>Gibco</td>
			<td>12491023</td>
			<td></td>
		</tr>
		<tr>
			<td>Albumin Bovine (cell culture grade)</td>
			<td>Serva</td>
			<td>47330</td>
			<td></td>
		</tr>
		<tr>
			<td>B-27 Supplement (50X), minus vitamin A</td>
			<td>Gibco</td>
			<td>12587010</td>
			<td></td>
		</tr>
		<tr>
			<td>Base moulds</td>
			<td>VWR</td>
			<td>720-1918</td>
			<td></td>
		</tr>
		<tr>
			<td>Buffer RLT</td>
			<td>Qiagen</td>
			<td>79216</td>
			<td></td>
		</tr>
		<tr>
			<td>Cassettes, Q Path Microtwin</td>
			<td>VWR</td>
			<td>720-2191</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell strainer, 40 &#181;m mesh, disposable</td>
			<td>Falcon</td>
			<td>352340</td>
			<td></td>
		</tr>
		<tr>
			<td>Cholera Toxin from Vibrio cholerae</td>
			<td>Sigma-Aldrich</td>
			<td>C8052</td>
			<td></td>
		</tr>
		<tr>
			<td>Deoxyribonuclease I from bovine pancreas</td>
			<td>Sigma-Aldrich</td>
			<td>D5025</td>
			<td></td>
		</tr>
		<tr>
			<td>D-glucose</td>
			<td>Merck</td>
			<td>108342</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethylsulfoxide (DMSO)</td>
			<td>Sigma-Aldrich</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM, powder, high glucose</td>
			<td>Gibco</td>
			<td>52100039</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf Safe-Lock Tubes, 1.5 mL</td>
			<td>Eppendorf</td>
			<td>30120086</td>
			<td></td>
		</tr>
		<tr>
			<td>Epredia SuperFrost Plus Adhesion slides</td>
			<td>Thermo Fisher Scientific</td>
			<td>J1800AMNZ</td>
			<td></td>
		</tr>
		<tr>
			<td>Epredia HistoStar Embedding Workstation, 220 to 240Vac</td>
			<td>Thermo Fisher Scientific</td>
			<td>12587976</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol Absolute 99.8+%</td>
			<td>Thermo Fisher Scientific</td>
			<td>10342652</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Sigma-Aldrich</td>
			<td>F7524</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMAX Supplement</td>
			<td>Gibco</td>
			<td>35050061</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H4034</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES Buffer Solution</td>
			<td>Gibco</td>
			<td>15630056</td>
			<td></td>
		</tr>
		<tr>
			<td>InSolution Y-27632</td>
			<td>Sigma-Aldrich</td>
			<td>688001</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine (200 mM)</td>
			<td>Gibco</td>
			<td>25030081</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel Growth Factor Reduced (GFR) Basement Membrane Matrix, LDEV-Free</td>
			<td>Corning</td>
			<td>15505739</td>
			<td></td>
		</tr>
		<tr>
			<td>Mr. Frosty Freezing Container</td>
			<td>Thermo Fisher Scientific</td>
			<td>5100-0001</td>
			<td></td>
		</tr>
		<tr>
			<td>N-2 Supplement (100X)</td>
			<td>Thermo Fisher Scientific</td>
			<td>17502048</td>
			<td></td>
		</tr>
		<tr>
			<td>N-Acetyl-L-cysteine</td>
			<td>Sigma-Aldrich</td>
			<td>A7250</td>
			<td></td>
		</tr>
		<tr>
			<td>Nunc Biobanking and Cell Culture Cryogenic Tubes</td>
			<td>Thermo Fisher Scientific</td>
			<td>375353</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde for synthesis (PFA)</td>
			<td>Merck</td>
			<td>818715</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS, pH 7.4</td>
			<td>Gibco</td>
			<td>10010023</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin G sodium salt</td>
			<td>Sigma-Aldrich</td>
			<td>P3032</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (10,000 U/mL)</td>
			<td>Gibco</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenol red</td>
			<td>Merck</td>
			<td>107241</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Chloride (KCl)</td>
			<td>Merck</td>
			<td>104936</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human EGF Protein, CF</td>
			<td>R&#38;D systems</td>
			<td>236-EG</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human FGF basic/FGF2/bFGF (157 aa) Protein</td>
			<td>R&#38;D systems</td>
			<td>234-FSE</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human FGF-10</td>
			<td>Peprotech</td>
			<td>100-26</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human IGF-1</td>
			<td>Peprotech</td>
			<td>100-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human IL-6</td>
			<td>Peprotech</td>
			<td>200-06</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human Noggin</td>
			<td>Peprotech</td>
			<td>120-10C</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human R-Spondin-1</td>
			<td>Peprotech</td>
			<td>120-38</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human/Murine FGF-8b</td>
			<td>Peprotech</td>
			<td>100-25</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Mouse Sonic Hedgehog/Shh (C25II) N-Terminus</td>
			<td>R&#38;D systems</td>
			<td>464-SH</td>
			<td></td>
		</tr>
		<tr>
			<td>RNeasy micro kit</td>
			<td>Qiagen</td>
			<td>74004</td>
			<td></td>
		</tr>
		<tr>
			<td>SB202190</td>
			<td>Sigma-Aldrich</td>
			<td>S7067</td>
			<td></td>
		</tr>
		<tr>
			<td>SeaKem LE Agarose</td>
			<td>Lonza</td>
			<td>50004</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride (NaCl)</td>
			<td>BDH</td>
			<td>102415K</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium di-Hydrogen Phosphate 1-hydrate</td>
			<td>PanReac-AppliChem</td>
			<td>A1047</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Hydrogen Carbonate (NaHCO3)</td>
			<td>Merck</td>
			<td>106329</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium-Pyruvate (C3H3NaO3)</td>
			<td>Sigma-Aldrich</td>
			<td>P5280</td>
			<td></td>
		</tr>
		<tr>
			<td>Stericup-GP, 0.22 &#181;m</td>
			<td>Millipore</td>
			<td>SCGPU02RE</td>
			<td></td>
		</tr>
		<tr>
			<td>Steriflip-GP Sterile Centrifuge Tube Top Filter Unit, 0.22 &#956;m</td>
			<td>Millipore</td>
			<td>SCGP00525</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile water</td>
			<td>Fresenius</td>
			<td>B230531</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptomycin sulfate salt</td>
			<td>Sigma-Aldrich</td>
			<td>S6501</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe, with BD Microlance needle with intradermal bevel, 26G</td>
			<td>BD Plastipak</td>
			<td>BDAM303176</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermo Scientific Excelsior ES Tissue Processor</td>
			<td>Thermo Scientific</td>
			<td>12505356</td>
			<td></td>
		</tr>
		<tr>
			<td>Titriplex III</td>
			<td>Merck</td>
			<td>108418</td>
			<td></td>
		</tr>
		<tr>
			<td>TrypL Express Enzyme (1X), phenol red</td>
			<td>Thermo Fisher Scientific</td>
			<td>12605028</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin inhibitor from Glycine max (soybean)</td>
			<td>Sigma-Aldrich</td>
			<td>T9003</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin solution 2.5 %</td>
			<td>Thermo Fisher Scientific</td>
			<td>15090046</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

After isolation and dissociation of the AL, the obtained single cells are seeded in ECM and grown in PitOM (Figure 1, Table 1). Figure 3A displays the cell culture and density at seeding (Day 0). Some small debris may be present (Figure 3A, white arrowheads), but will disappear at passaging. Fourteen days after seeding, the AL-derived organoids are fully developed (Figure 3A). The organ...

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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.

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, ...

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3.8K Views.  KU Leuven (University of Leuven). 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.

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

Epigenetic Conversion as a Safe and Simple Method to Obtain Insulin-secreting Cells from Adult Skin Fibroblasts

Regenerative medicine requires new, fully functional cells that are delivered to patients in order to repair degenerated or damaged tissues. When such cells are not readily available, they can be obtained using different approaches that include, among the many, reprogramming and trans-differentiation, with advantages and limitations that are specific of the different techniques. Here a new strategy for the conversion of an adult mature fibroblast into an insulin-secreting cell, arbitrarily designated as epigenetic converted cells (EpiCC), is described. The method has been developed, based on the increasing understanding of the mechanisms controlling epigenetic regulation of cell fate and differentiation. In particular, the first step uses an epigenetic modifier, namely 5-aza-cytidine, to drive adult cells into a "highly permissive" state. It then takes advantage of this brief and reversible window of epigenetic plasticity, to re-address cells toward a different lineage. The approach is designated "epigenetic cell conversion". It is a simple and robust way to obtain an efficient, controlled and stable cellular inter-lineage switch. Since the protocol does not involve the use of any gene transfection, it is free of viral vectors and does not involve a stable pluripotent state, it is highly promising for translational medicine applications.

Here, a new method that allows the conversion of adult skin fibroblasts into insulin-secreting cells is presented. This technique is based on epigenetic conversion, does not involve the use of retroviral vectors nor the acquisition of a stable pluripotent state. It is therefore highly promising for translational medicine applications.

Regenerative medicine requires new, fully functional cells that are delivered to patients in order to repair degenerated or damaged tissues. When such ...

Effective Isolation of Functional Islets from Neonatal Mouse Pancreas

Perfusion-based islet-isolation protocols from large mammalian pancreata are well established. Such protocols are readily conducted in many laboratories due to the large size of the pancreatic duct that allows for ready collagenase injection and subsequent tissue perfusion. In contrast, islet isolation from small pancreata, like that of neonatal mice, is challenging because perfusion is not readily achievable in the small pancreata. Here we describe a detailed simple procedure that recovers substantial numbers of islets from newly born mice with visual assistance. Freshly dissected whole pancreata were digested with 0.5 mg/mL collagenase IV dissolved in Hanks&#39; Balanced Salt Solution (HBSS) at 37 &#176;C, in microcentrifuge tubes. Tubes were tapped regularly to aid tissue dispersal. When most of the tissue was dispersed to small clusters around 1 mm, lysates were washed three to four times with culture media with 10% fetal bovine serum (FBS). Islet clusters, devoid of recognizable acinar tissues, can then be recovered under dissecting stereoscope. This method recovers 20 - 80 small- to large-sized islets per pancreas of newly born mouse. These islets are suitable for most conceivable downstream assays, including insulin secretion, gene expression, and culture. An example of insulin secretion assay is presented to validate the isolation process. The genetic background and degree of digestion are the largest factors determining the yield. Freshly made collagenase solution with high activity is preferred, as it aids in endocrine-exocrine isolation. The presence of cations [calcium (Ca2+) and magnesium (Mg2+)] in all solutions and fetal bovine serum in the wash/picking media are necessary for good yield of islets with proper integrity. A dissecting scope with good contrast and magnification will also help.

We describe a protocol herein for isolating intact islets from neonatal mice. Pancreata were partially digested with collagenase, followed by washing and hand picking. 20 - 80 islet clusters can be obtained per pancreas from newly born mice, which are suitable for several islet studies.

Perfusion-based islet-isolation protocols from large mammalian pancreata are well established. Such protocols are readily conducted in many laboratories ...

Dissection and Coronal Slice Preparation of Developing Mouse Pituitary Gland

The pituitary gland or hypophysis is an important endocrine organ secreting hormones essential for homeostasis. It consists of two glands with separate embryonic origins and functions &#8212; the neurohypophysis and the adenohypophysis. The developing mouse pituitary gland is tiny and delicate with an elongated oval shape. A coronal section is preferred to display both the adenohypophysis and neurohypophysis in a single slice of the mouse pituitary.
The goal of this protocol is to achieve proper pituitary coronal sections with well-preserved tissue architectures from developing mice. In this protocol, we describe in detail how to dissect and process pituitary glands properly from developing mice. First, mice are fixed by transcardial perfusion of formaldehyde prior to dissection. Then three different dissecting techniques are applied to obtain intact pituitary glands depending on the age of mice. For fetal mice aged embryonic days (E) 17.5 - 18.5 and neonates up to 4 days, the entire sella regions including the sphenoid bone, gland, and trigeminal nerves are dissected. For pups aged postnatal days (P) 5 - 14, the pituitary glands connected with trigeminal nerves are dissected as a whole. For mice over 3 weeks old, the pituitary glands are carefully dissected free from the surrounding tissues. We also display how to embed the pituitary glands in a proper orientation by using the surrounding tissues as landmarks to obtain satisfying coronal sections. These methods are useful in analyzing histological and developmental features of pituitary glands in developing mice.

We present a protocol to dissect pituitary glands and prepare pituitary coronal sections from developing mice.

The pituitary gland or hypophysis is an important endocrine organ secreting hormones essential for homeostasis. It consists of two glands with separate ...

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell types include enzyme-secreting acinar cells, an arborized ductal system responsible for the transportation of enzymes to the gut, and hormone-producing endocrine cells. 
Endocrine beta-cells are the sole cell type in the body that produce insulin to lower blood glucose levels. Diabetes, a disease characterized by a loss or the dysfunction of beta-cells, is reaching epidemic proportions. Thus, it is essential to establish protocols to investigate beta-cell development that can be used for screening purposes to derive the drug and cell-based therapeutics. While the experimental investigation of mouse development is essential, in vivo studies are laborious and time-consuming. Cultured cells provide a more convenient platform for screening; however, they are unable to maintain the cellular diversity, architectural organization, and cellular interactions found in vivo. Thus, it is essential to develop new tools to investigate pancreatic organogenesis and physiology.
Pancreatic epithelial cells develop in the close association with mesenchyme from the onset of organogenesis as cells organize and differentiate into the complex, physiologically competent adult organ. The pancreatic mesenchyme provides important signals for the endocrine development, many of which are not well understood yet, thus difficult to recapitulate during the in vitro culture. Here, we describe a protocol to culture three-dimensional, cellular complex mouse organoids that retain mesenchyme, termed pancreatoids. The e10.5 murine pancreatic bud is dissected, dissociated, and cultured in a scaffold-free environment. These floating cells self-assemble with mesenchyme enveloping the developing pancreatoid and a robust number of endocrine beta-cells developing along with the acinar and the duct cells. This system can be used to study the cell fate determination, structural organization, and morphogenesis, cell-cell interactions during organogenesis, or for the drug, small molecule, or genetic screening.

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

Here, we present a protocol to generate insulin expressing 3D murine pancreatoids from free-floating e10.5 dissociated pancreatic progenitors and the associated mesenchyme.

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell ...

Analysis of Cardiac Chamber Development During Mouse Embryogenesis Using Whole Mount Epifluorescence

The goal of this protocol is to describe a method for the dissection of mouse embryos and visualization of embryonic mouse ventricular chambers during heart development using ventricular specific fluorescent reporter knock-in mice (MLC-2v-tdTomato mice). Heart development involves a linear heart tube formation, the heart tube looping, and four chamber septation. These complex processes are highly conserved in all vertebrates. The mouse embryonic heart has been widely used for heart developmental studies. However, due to their extremely small size, dissecting mouse embryonic hearts is technically challenging. In addition, visualization of cardiac chamber formation often needs in situ hybridization, beta-galactosidase staining using LacZ reporter mice, or immunostaining of sectioned embryonic hearts. Here, we describe how to dissect mouse embryonic hearts and directly visualize ventricular chamber formation of MLC-2v-tdTomato mice using whole mount epifluorescent microscopy. With this method, it is possible to directly examine heart tube formation and looping, and four chamber formation without further experimental manipulation of mouse embryos. Although the MLC-2v-tdTomato reporter knock-in mouse line is used in this protocol as an example, this protocol can be applied to other heart-specific fluorescent reporter transgenic mouse lines.

We present the protocols to examine mouse heart development using whole mount epifluorescent microscopy on mouse embryos dissected from ventricular specific MLC-2v-tdTomato reporter knock-in mice. This method allows us to directly visualize each stage of the ventricular formation during mouse heart development without labor-intensive histochemical methods.

The goal of this protocol is to describe a method for the dissection of mouse embryos and visualization of embryonic mouse ventricular chambers during ...

Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C (UV-C) Damage

The ocular surface is subjected to regular wear and tear due to various environmental factors. Exposure to UV-C radiation constitutes an occupational health hazard. Here, we demonstrate the exposure of primary stem cells from the mouse ocular surface to UV-C radiation. Reactive oxygen species (ROS) formation is the readout of the extent of oxidative stress/damage. In an experimental in vitro setting, it is also essential to assess the percentage of dead cells generated due to oxidative stress. In this article, we will demonstrate the 2&#39;,7&#39;-Dichlorofluoresceindiacetate (DCFDA) staining of UV-C exposed mouse primary ocular surface stem cells and their quantification based on the fluorescent images of DCFDA staining. DCFDA staining directly corresponds to ROS generation. We also demonstrate the quantification of dead and live cells by simultaneous staining with propidium iodide (PI) and Hoechst 3332 respectively and the percentage of DCFDA (ROS positive) and PI positive cells.

Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C UV-C Damage

This protocol demonstrates the simultaneous detection of reactive oxygen species (ROS), live cells, and dead cells in live primary cultures from mouse ocular surface cells. 2&#39;,7&#39;-Dichlorofluoresceindiacetate, propidium iodide, and Hoechst staining are used to assess the ROS, dead cells, and live cells, respectively, followed by imaging and analysis.

The ocular surface is subjected to regular wear and tear due to various environmental factors. Exposure to UV-C radiation constitutes an occupational ...

A Static Self-Directed Method for Generating Brain Organoids from Human Embryonic Stem Cells

Human brain organoids differentiated from embryonic stem cells offer the unique opportunity to study complicated interactions of multiple cell types in a three-dimensional system. Here we present a relatively straightforward and inexpensive method that yields brain organoids. In this protocol human pluripotent stem cells are broken into small clusters instead of single cells and grown in basic media without a heterologous basement membrane matrix or exogenous growth factors, allowing the intrinsic developmental cues to shape the organoid&#39;s growth. This simple system produces a diversity of brain cell types including glial and microglial cells, stem cells, and neurons of the forebrain, midbrain, and hindbrain. Organoids generated from this protocol also display hallmarks of appropriate temporal and spatial organization demonstrated by brightfield images, histology, immunofluorescence and real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). Because these organoids contain cell types from various parts of the brain, they can be utilized for studying a multitude of diseases. For example, in a recent paper we demonstrated the use of organoids generated from this protocol for studying the effects of hypoxia on the human brain. This approach can be used to investigate an array of otherwise difficult to study conditions such as neurodevelopmental handicaps, genetic disorders, and neurologic diseases.

This protocol was generated as a means to produce brain organoids in a simplified, low cost manner without exogenous growth factors or basement membrane matrix while still maintaining the diversity of brain cell types and many features of cellular organization.

Human brain organoids differentiated from embryonic stem cells offer the unique opportunity to study complicated interactions of multiple cell types in a ...

Preparation of Small RNA Libraries for Sequencing from Early Mouse Embryos

MicroRNAs (miRNAs) are important for the complex regulation of cell fate decisions and developmental timing. In vivo studies of the contribution of miRNAs during early development are technically challenging due to the limiting cell number. Moreover, many approaches require a miRNA of interest to be defined in assays such as northern blotting, microarray, and qPCR. Therefore, the expression of many miRNAs and their isoforms have not been studied during early development. Here, we demonstrate a protocol for small RNA sequencing of sorted cells from early mouse embryos to enable relatively unbiased profiling of miRNAs in early populations of neural crest cells. We overcome the challenges of low cell input and size selection during library preparation using amplification and gel-based purification. We identify embryonic age as a variable accounting for variation between replicates and stage-matched mouse embryos must be used to accurately profile miRNAs in biological replicates. Our results suggest that this method can be broadly applied to profile the expression of miRNAs from other lineages of cells. In summary, this protocol can be used to study how miRNAs regulate developmental programs in different cell lineages of the early mouse embryo.

We describe a technique for profiling microRNAs in early mouse embryos. This protocol overcomes the challenge of low cell input and small RNA enrichment. This assay can be used to analyze changes in miRNA expression over time in different cell lineages of the early mouse embryo.

MicroRNAs (miRNAs) are important for the complex regulation of cell fate decisions and developmental timing. In vivo studies of the contribution of miRNAs ...

In vitro Neuromuscular Junction Induced from Human Induced Pluripotent Stem Cells

The neuromuscular junction (NMJ) is a specialized synapse that transmits action potentials from the motor neuron to skeletal muscle for mechanical movement. The architecture of the NMJ structure influences the functions of the neuron, the muscle and the mutual interaction. Previous studies have reported many strategies by co-culturing the motor neurons and myotubes to generate NMJ in vitro with complex induction process and long culture period but have struggled to recapitulate mature NMJ morphology and function. Our in vitro NMJ induction system is constructed by differentiating human iPSC in a single culture dish. By switching the myogenic and neurogenic induction medium for induction, the resulting NMJ contained pre- and post- synaptic components, including motor neurons, skeletal muscle and Schwann cells in the one month culture. The functional assay of NMJ also showed that the myotubes contraction can be triggered by Ca++ then inhibited by curare, an acetylcholine receptor (AChR) inhibitor, in which the stimulating signal is transmitted through NMJ. This simple and robust approach successfully derived the complex structure of NMJ with functional connectivity. This in vitro human NMJ, with its integrated structures and function, has promising potential for studying pathological mechanisms and compound screening.

Here we provide a protocol to generate in vitro NMJs from human induced pluripotent stem cells (iPSCs). This method can induce NMJs with mature morphology and function in 1 month in a single well. The resulting NMJs could potentially be used to model related diseases, to study pathological mechanisms or to screen drug compounds for therapy.

The neuromuscular junction (NMJ) is a specialized synapse that transmits action potentials from the motor neuron to skeletal muscle for mechanical ...

Generation and Culture of Lingual Organoids Derived from Adult Mouse Taste Stem Cells

The sense of taste is mediated by taste buds on the tongue, which are composed of rapidly renewing taste receptor cells (TRCs). This continual turnover is powered by local progenitor cells and renders taste function prone to disruption by a multitude of medical treatments, which in turn severely impacts the quality of life. Thus, studying this process in the context of drug treatment is vital to understanding if and how taste progenitor function and TRC production are affected. Given the ethical concerns and limited availability of human taste tissue, mouse models, which have a taste system similar to humans, are commonly used. Compared to in vivo methods, which are time-consuming, expensive, and not amenable to high throughput studies, murine lingual organoids can enable experiments to be run rapidly with many replicates and fewer mice. Here, previously published protocols have been adapted and a standardized method for generating taste organoids from taste progenitor cells isolated from the circumvallate papilla (CVP) of adult mice is presented. Taste progenitor cells in the CVP express LGR5 and can be isolated via EGFP fluorescence-activated cell sorting (FACS) from mice carrying an Lgr5EGFP-IRES-CreERT2 allele. Sorted cells are plated onto a matrix gel-based 3D culture system and cultured for 12 days. Organoids expand for the first 6 days of the culture period via proliferation and then enter a differentiation phase, during which they generate all three taste cell types along with non-taste epithelial cells. Organoids can be harvested upon maturation at day 12 or at any time during the growth process for RNA expression and immunohistochemical analysis. Standardizing culture methods for production of lingual organoids from adult stem cells will improve reproducibility and advance lingual organoids as a powerful drug screening tool in the fight to help patients experiencing taste dysfunction.

The protocol presents a method for culturing and processing lingual organoids derived from taste stem cells isolated from the posterior taste papilla of adult mice.

The sense of taste is mediated by taste buds on the tongue, which are composed of rapidly renewing taste receptor cells (TRCs). This continual turnover is ...