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><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/F12</td><td>Gibco</td><td>12634010</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 &amp;micro;m mesh, disposable</td><td>Falcon</td><td>352340</td><td></td></tr><tr><td>Cholera Toxin from &lt;em&gt;Vibrio &lt;/em&gt;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&amp;amp;D systems</td><td>236-EG</td><td></td></tr><tr><td>Recombinant Human FGF basic/FGF2/bFGF (157 aa) Protein</td><td>R&amp;amp;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&amp;amp;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 &amp;micro;m</td><td>Millipore</td><td>SCGPU02RE</td><td></td></tr><tr><td>Steriflip-GP Sterile Centrifuge Tube Top Filter Unit, 0.22 &amp;mu;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>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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Research

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Developmental Biology

3.9K 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

Prostate Organoid Cultures as Tools to Translate Genotypes and Mutational Profiles to Pharmacological Responses

Presented here is a protocol to study pharmacodynamics, stem cell potential, and cancer differentiation in prostate epithelial organoids. Prostate organoids are androgen responsive, three-dimensional (3D) cultures grown in a defined medium that resembles the prostatic epithelium. Prostate organoids can be established from wild-type and genetically engineered mouse models, benign human tissue, and advanced prostate cancer. Importantly, patient derived organoids closely resemble tumors in genetics and in vivo tumor biology. Moreover, organoids can be genetically manipulated using CRISPR/Cas9 and shRNA systems. These controlled genetics make the organoid culture attractive as a platform for rapidly testing the effects of genotypes and mutational profiles on pharmacological responses. However, experimental protocols must be specifically adapted to the 3D nature of organoid cultures to obtain reproducible results. Described here are detailed protocols for performing seeding assays to determine organoid formation capacity. Subsequently, this report shows how to perform drug treatments and analyze pharmacological response via viability measurements, protein isolation, and RNA isolation. Finally, the protocol describes how to prepare organoids for xenografting and subsequent in vivo growth assays using subcutaneous grafting. These protocols yield highly reproducible data and are widely applicable to 3D culture systems.

Presented here is a protocol to study pharmacological responses in prostate epithelial organoids. Organoids closely resemble in vivo biology and recapitulate patient genetics, making them attractive model systems. Prostate organoids can be established from wildtype prostates, genetically engineered mouse models, benign human tissue, and advanced prostate cancer.

Presented here is a protocol to study pharmacodynamics, stem cell potential, and cancer differentiation in prostate epithelial organoids. Prostate ...

Cancer Research

Generation of Standardized and Reproducible Forebrain-type Cerebral Organoids from Human Induced Pluripotent Stem Cells

The human cortex is highly expanded and exhibits a complex structure with specific functional areas, providing higher brain function, such as cognition. Efforts to study human cerebral cortex development have been limited by the availability of model systems. Translating results from rodent studies to the human system is restricted by species differences and studies on human primary tissues are hampered by a lack of tissue availability as well as ethical concerns. Recent development in human pluripotent stem cell (PSC) technology include the generation of three-dimensional (3D) self-organizing organotypic culture systems, which mimic to a certain extent human-specific brain development in vitro. Currently, various protocols are available for the generation of either whole brain or brain-region specific organoids. The method for the generation of homogeneous and reproducible forebrain-type organoids from induced PSC (iPSC), which we previously established and describe here, combines the intrinsic ability of PSC to self-organize with guided differentiation towards the anterior neuroectodermal lineage and matrix embedding to support the formation of a continuous neuroepithelium. More specifically, this protocol involves: (1) the generation of iPSC aggregates, including the conversion of iPSC colonies to a confluent monolayer culture; (2) the induction of anterior neuroectoderm; (3) the embedding of neuroectodermal aggregates in a matrix scaffold; (4) the generation of forebrain-type organoids from neuroectodermal aggregates; and (5) the fixation and validation of forebrain-type organoids. As such, this protocol provides an easily applicable system for the generation of standardized and reproducible iPSC-derived cortical tissue structures in vitro.

Cerebral organoids represent a new model system to investigate early human brain development in vitro. This article provides the detailed methodology to efficiently generate homogeneous dorsal forebrain-type organoids from human induced pluripotent stem cells including critical characterization and validation steps.

The human cortex is highly expanded and exhibits a complex structure with specific functional areas, providing higher brain function, such as cognition. ...

Three-Dimensional Imaging of Organoids to Study Primary Ciliogenesis During ex vivo Organogenesis

Organoids are stem cell-derived three-dimensional structures that reproduce ex vivo the complex architecture and physiology of organs. Thus, organoids represent useful models to study the mechanisms that control stem cell self-renewal and differentiation in mammals, including primary ciliogenesis and ciliary signaling. Primary ciliogenesis is the dynamic process of assembling the primary cilium, a key cell signaling center that controls stem cell self-renewal and/or differentiation in various tissues. Here we present a comprehensive protocol for the immunofluorescence staining of cell lineage and primary cilia markers, in whole-mount mouse mammary organoids, for light sheet microscopy. We describe the microscopy imaging method and an image processing technique for the quantitative analysis of primary cilium assembly and length in organoids. This protocol enables a precise analysis of primary cilia in complex three-dimensional structures at the single cell level. This method is applicable for immunofluorescence staining and imaging of primary cilia and ciliary signaling in mammary organoids derived from normal and genetically modified stem cells, from healthy and pathological tissues, to study the biology of the primary cilium in health and disease.

Three-Dimensional Imaging of Organoids to Study Primary Ciliogenesis During ex vivo Organogenesis

Stem cell-derived organoids facilitate the analysis of molecular and cellular processes that regulate stem cell self-renewal and differentiation during organogenesis in mammalian tissues. Here we present a protocol for the analysis of the biology of the primary cilium in mouse mammary organoids.

Organoids are stem cell-derived three-dimensional structures that reproduce ex vivo the complex architecture and physiology of organs. Thus, organoids ...

Genetics

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 ...

Generation of Organoids from Mouse Extrahepatic Bile Ducts

Cholangiopathies, which affect extrahepatic bile ducts (EHBDs), include biliary atresia, primary sclerosing cholangitis, and cholangiocarcinoma. They have no effective therapeutic options. Tools to study EHBD are very limited. Our purpose was to develop an organ-specific, versatile, adult stem cell-derived, preclinical cholangiocyte model that can be easily generated from wild type and genetically engineered mice. Thus, we report on the novel technique of developing an EHBD organoid (EHBDO) culture system from adult mouse EHBDs. The model is cost-efficient, able to be readily analyzed, and has multiple downstream applications. Specifically, we describe the methodology of mouse EHBD isolation and single cell dissociation, organoid culture initiation, propagation, and long-term maintenance and storage. This manuscript also describes EHBDO processing for immunohistochemistry, fluorescent microscopy, and mRNA abundance quantitation by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). This protocol has significant advantages in addition to producing EHBD-specific organoids. The use of a conditioned medium from L-WRN cells significantly reduces the cost of this model. The use of mouse EHBDs provides almost unlimited tissue for culture generation, unlike human tissue. Generated mouse EHBDOs contain a pure population of epithelial cells with markers of endodermal progenitor and differentiated biliary cells. Cultured organoids maintain homogenous morphology through multiple passages and can be recovered after a long-term storage period in liquid nitrogen. The model allows for the study of biliary progenitor cell proliferation, can be manipulated pharmacologically, and may be generated from genetically engineered mice. Future studies are needed to optimize culture conditions in order to increase plating efficiency, evaluate functional cell maturity, and direct cell differentiation. Development of co-culture models and a more biologically neutral extracellular matrix are also desirable.

This protocol describes the production of a mouse extrahepatic bile duct 3-dimensional organoid system. These biliary organoids can be maintained in culture to study cholangiocyte biology. Biliary organoids express markers of both progenitor and biliary cells and are composed of polarized epithelial cells.

Cholangiopathies, which affect extrahepatic bile ducts (EHBDs), include biliary atresia, primary sclerosing cholangitis, and cholangiocarcinoma. They have ...

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 ...

Extra Cellular Matrix-Based and Extra Cellular Matrix-Free Generation of Murine Testicular Organoids

Testicular organoids provide a tool for studying testicular development, spermatogenesis, and endocrinology in vitro. Several methods have been developed in order to create testicular organoids. Many of these methods rely upon extracellular matrix (ECM) to promote de novo tissue assembly, however, there are differences between methods in terms of biomimetic morphology and function of tissues. Moreover, there are few direct comparisons of published methods. Here, a direct comparison is made by studying differences in organoid generation protocols, with provided outcomes. Four archetypal generation methods: (1) 2D ECM-free, (2) 2D ECM, (3) 3D ECM-free, and (4) 3D ECM culture are described. Three primary benchmarks were used to assess the testicular organoid generation. These are cellular self-assembly, inclusion of major cell types (Sertoli, Leydig, germ, and peritubular cells), and appropriately compartmentalized tissue architecture. Of the four environments tested, 2D ECM and 3D ECM-free cultures generated organoids with internal morphologies most similar to native testes, including the de novo compartmentalization of tubular versus interstitial cell types, the development of tubule-like-structures, and an established long-term endocrine function. All methods studied utilized unsorted, primary murine testicular cell suspensions and used commonly accessible culture resources. These testicular organoid generation techniques provide a highly accessible and reproducible toolkit for research initiatives into testicular organogenesis and physiology in vitro.

Here, four methods for generating testicular organoids from primary neonatal murine testicular cells are described i.e., extracellular matrix (ECM) and ECM-free 2D and 3D culture environments. These techniques have multiple research applications and are especially useful for studying testicular development and physiology in vitro.

Testicular organoids provide a tool for studying testicular development, spermatogenesis, and endocrinology in vitro. Several methods have been developed ...

2D and 3D Human Induced Pluripotent Stem Cell-Based Models to Dissect Primary Cilium Involvement during Neocortical Development

Primary cilia (PC) are non-motile dynamic microtubule-based organelles that protrude from the surface of most mammalian cells. They emerge from the older centriole during the G1/G0 phase of the cell cycle, while they disassemble as the cells re-enter the cell cycle at the G2/M phase boundary. They function as signal hubs, by detecting and transducing extracellular signals crucial for many cell processes. Similar to most cell types, all neocortical neural stem and progenitor cells (NSPCs) have been shown harboring a PC allowing them to sense and transduce specific signals required for the normal cerebral cortical development. Here, we provide detailed protocols to generate and characterize two-dimensional (2D) and three-dimensional (3D) cell-based models from human induced pluripotent stem cells (hIPSCs) to further dissect the involvement of PC during neocortical development. In particular, we present protocols to study the PC biogenesis and function in 2D neural rosette-derived NSPCs including the transduction of the Sonic Hedgehog (SHH) pathway. To take advantage of the three-dimensional (3D) organization of cerebral organoids, we describe a simple method for 3D imaging of in toto immunostained cerebral organoids. After optical clearing, rapid acquisition of entire organoids allows detection of both centrosomes and PC on neocortical progenitors and neurons of the whole organoid. Finally, we detail the procedure for immunostaining and clearing of thick free-floating organoid sections preserving a significant degree of 3D spatial information and allowing for the high-resolution acquisition required for the detailed qualitative and quantitative analysis of PC biogenesis and function.

We present detailed protocols for the generation and characterization of 2D and 3D human induced pluripotent stem cell (hIPSC)-based models of neocortical development as well as complementary methodologies enabling qualitative and quantitative analysis of primary cilium (PC) biogenesis and function.

Primary cilia (PC) are non-motile dynamic microtubule-based organelles that protrude from the surface of most mammalian cells. They emerge from the older ...

Generation of 3D Midbrain Organoids from Human-Induced Pluripotent Stem Cells

The development of midbrain organoids (MOs) from human pluripotent stem cells (hPSCs) represents a significant advancement in understanding brain development, facilitating precise disease modeling, and advancing therapeutic research. This protocol outlines a method for generating midbrain-specific organoids using induced pluripotent stem cells (iPSCs), employing a strategic differentiation approach. Key techniques include dual-SMAD inhibition to suppress SMAD signaling, administration of fibroblast growth factor 8b (FGF-8b), and activation of the Sonic Hedgehog pathway using the agonist purmorphamine, guiding iPSCs towards a midbrain fate.
The organoids produced by this method achieve diameters up to 2 mm and incorporate a diverse array of neuroepithelial cell types, reflecting the midbrain&#39;s inherent cellular diversity. Validation of these organoids as authentic midbrain structures involves the expression of midbrain-specific markers, confirming their identity. A notable outcome of this methodology is the effective differentiation of iPSCs into dopaminergic neurons, which are characteristic of the midbrain.
The significance of this protocol lies in its ability to produce functionally mature, midbrain-specific organoids that closely replicate essential aspects of the midbrain, offering a valuable model for in-depth exploration of midbrain developmental processes and the pathophysiology of related disorders such as Parkinson&#39;s disease. Thus, this protocol serves as a crucial resource for researchers seeking to enhance our understanding of the human brain and develop new treatments for neurodegenerative diseases, making it an indispensable tool in the field of neurological research.

This protocol describes a novel method for creating 3D midbrain organoids from human induced pluripotent stem cells, guiding their formation to mimic native midbrain tissue, thereby aiding in the study of development and disorders.

The development of midbrain organoids (MOs) from human pluripotent stem cells (hPSCs) represents a significant advancement in understanding brain ...

In Vitro Pancreas Organogenesis from Dispersed Mouse Embryonic Progenitors

The pancreas is an essential organ that regulates glucose homeostasis and secretes digestive enzymes. Research on pancreas embryogenesis has led to the development of protocols to produce pancreatic cells from stem cells 1. The whole embryonic organ can be cultured at multiple stages of development 2-4. These culture methods have been useful to test drugs and to image developmental processes. However the expansion of the organ is very limited and morphogenesis is not faithfully recapitulated since the organ flattens. 
We propose three-dimensional (3D) culture conditions that enable the efficient expansion of dissociated mouse embryonic pancreatic progenitors. By manipulating the composition of the culture medium it is possible to generate either hollow spheres, mainly composed of pancreatic progenitors expanding in their initial state, or, complex organoids which progress to more mature expanding progenitors and differentiate into endocrine, acinar and ductal cells and which spontaneously self-organize to resemble the embryonic pancreas. 
We show here that the in vitro process recapitulates many aspects of natural pancreas development. This culture system is suitable to investigate how cells cooperate to form an organ by reducing its initial complexity to few progenitors. It is a model that reproduces the 3D architecture of the pancreas and that is therefore useful to study morphogenesis, including polarization of epithelial structures and branching. It is also appropriate to assess the response to mechanical cues of the niche such as stiffness and the effects on cell&#180;s tensegrity.

In Vitro Pancreas Organogenesis from Dispersed Mouse Embryonic Progenitors

The three-dimensional culture method described in this protocol recapitulates pancreas development from dispersed embryonic mouse pancreas progenitors, including their substantial expansion, differentiation and morphogenesis into a branched organ. This method is amenable to imaging, functional interference and manipulation of the niche.

The pancreas is an essential organ that regulates glucose homeostasis and secretes digestive enzymes. Research on pancreas embryogenesis has led to the ...

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

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Chapters in this video

Prostate Organoid Cultures as Tools to Translate Genotypes and Mutational Profiles to Pharmacological Responses

Generation of Standardized and Reproducible Forebrain-type Cerebral Organoids from Human Induced Pluripotent Stem Cells

Three-Dimensional Imaging of Organoids to Study Primary Ciliogenesis During ex vivo Organogenesis

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

Generation of Organoids from Mouse Extrahepatic Bile Ducts

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

Extra Cellular Matrix-Based and Extra Cellular Matrix-Free Generation of Murine Testicular Organoids

2D and 3D Human Induced Pluripotent Stem Cell-Based Models to Dissect Primary Cilium Involvement during Neocortical Development

Generation of 3D Midbrain Organoids from Human-Induced Pluripotent Stem Cells

In Vitro Pancreas Organogenesis from Dispersed Mouse Embryonic Progenitors