Name: development of organoids from mouse pituitary as in vitro model to explore pituitary stem cell biology
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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...

<ol>
	<li>Melmed, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The pituitary. 3rd ed. , 1 (2011).</li><li>Chen, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+adult+pituitary+contains+a+cell+population+displaying+stem/progenitor+cell+and+early-embryonic+characteristics.">The adult pituitary contains a cell population displaying stem/progenitor cell and early-embryonic characteristics.</a> Endocrinology. 146 (9), 3985-3998 (2005).</li><li>Chen, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pituitary+progenitor+cells+tracked+down+by+side+population+dissection.">Pituitary progenitor cells tracked down by side population dissection.</a> Stem Cells. 27 (5), 1182-1195 (2009).</li><li>Fauquier, T., Rizzoti, K., Dattani, M., Lovell-Badge, R., Robinson, I. C. A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SOX2-expressing+progenitor+cells+generate+all+of+the+major+cell+types+in+the+adult+mouse+pituitary+gland.">SOX2-expressing progenitor cells generate all of the major cell types in the adult mouse pituitary gland.</a> Proceedings of the National Academy of Sciences of the United States of America. 105 (8), 2907-2912 (2008).</li><li>Rizzoti, K., Akiyama, H., Lovell-Badge, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mobilized+adult+pituitary+stem+cells+contribute+to+endocrine+regeneration+in+response+to+physiological+demand.">Mobilized adult pituitary stem cells contribute to endocrine regeneration in response to physiological demand.</a> Cell Stem Cell. 13 (4), 419-432 (2013).</li><li>Andoniadou, C. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sox2++stem/progenitor+cells+in+the+adult+mouse+pituitary+support+organ+homeostasis+and+have+tumor-inducing+potential.">Sox2+ stem/progenitor cells in the adult mouse pituitary support organ homeostasis and have tumor-inducing potential.</a> Cell Stem Cell. 13 (4), 433-445 (2013).</li><li>Nys, C., Vankelecom, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pituitary+disease+and+recovery:+How+are+stem+cells+involved.">Pituitary disease and recovery: How are stem cells involved.</a> Molecular and Cellular Endocrinology. 525 (4), 111176 (2021).</li><li>Schneider, H. J., Aimaretti, G., Kreitschmann-Andermahr, I., Stalla, G. K., Ghigo, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypopituitarism.">Hypopituitarism.</a> Lancet. 369 (9571), 1461-1470 (2007).</li><li>Fu, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+adult+pituitary+shows+stem/progenitor+cell+activation+in+response+to+injury+and+is+capable+of+regeneration.">The adult pituitary shows stem/progenitor cell activation in response to injury and is capable of regeneration.</a> Endocrinology. 153 (7), 3224-3235 (2012).</li><li>Willems, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regeneration+in+the+pituitary+after+cell-ablation+injury:+time-related+aspects+and+molecular+analysis.">Regeneration in the pituitary after cell-ablation injury: time-related aspects and molecular analysis.</a> Endocrinology. 157 (2), 705-721 (2016).</li><li>Gremeaux, L., Fu, Q., Chen, J., Vankelecom, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activated+phenotype+of+the+pituitary+stem/progenitor+cell+compartment+during+the+early-postnatal+maturation+phase+of+the+gland.">Activated phenotype of the pituitary stem/progenitor cell compartment during the early-postnatal maturation phase of the gland.</a> Stem Cells and Development. 21 (5), 801-813 (2012).</li><li>Zhu, X., Tollkuhn, J., Taylor, H., Rosenfeld, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Notch-dependent+pituitary+SOX2++stem+cells+exhibit+a+timed+functional+extinction+in+regulation+of+the+postnatal+gland.">Notch-dependent pituitary SOX2+ stem cells exhibit a timed functional extinction in regulation of the postnatal gland.</a> Stem Cell Reports. 5 (6), 1196-1209 (2015).</li><li>Russell, J. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pituitary+stem+cells+produce+paracrine+WNT+signals+to+control+the+expansion+of+their+descendant+progenitor+cells.">Pituitary stem cells produce paracrine WNT signals to control the expansion of their descendant progenitor cells.</a> eLife. 10 (1), 59142 (2021).</li><li>Vennekens, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interleukin-6+is+an+activator+of+pituitary+stem+cells+upon+local+damage,+a+competence+quenched+in+the+aging+gland.">Interleukin-6 is an activator of pituitary stem cells upon local damage, a competence quenched in the aging gland.</a> Proceedings of the National Academy of Sciences of the United States of America. 118 (25), 2100052118 (2021).</li><li>Mertens, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pituitary+tumors+contain+a+side+population+with+tumor+stem+cell-associated+characteristics.">Pituitary tumors contain a side population with tumor stem cell-associated characteristics.</a> Endocrine-Related Cancer. 22 (4), 481-504 (2015).</li><li>Laporte, E., Vennekens, A., Vankelecom, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pituitary+remodeling+throughout+life:+are+resident+stem+cells+involved.">Pituitary remodeling throughout life: are resident stem cells involved.</a> Frontiers in Endocrinology. 11 (1), 604519 (2021).</li><li>Yoshida, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+adult+pituitary+stem/progenitor+cell+clusters+located+in+the+parenchyma+of+the+rat+anterior+lobe.">Isolation of adult pituitary stem/progenitor cell clusters located in the parenchyma of the rat anterior lobe.</a> Stem Cell Research. 17 (2), 318-329 (2016).</li><li>Cox, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organoids+from+pituitary+as+novel+research+model+to+study+pituitary+stem+cell+biology.">Organoids from pituitary as novel research model to study pituitary stem cell biology.</a> Journal of Endocrinology. 240 (2), 287-308 (2019).</li><li>Denef, C., Hautekeete, E., De Wolf, A., Vanderschueren, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pituitary+basophils+from+immature+male+and+female+rats:+distribution+of+gonadotrophs+and+thyrotrophs+as+studied+by+unit+gravity+sedimentation.">Pituitary basophils from immature male and female rats: distribution of gonadotrophs and thyrotrophs as studied by unit gravity sedimentation.</a> Endocrinology. 130 (3), 724-735 (1978).</li><li>Vander Schueren, B., Denef, C., Cassiman, J. 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I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNAs+miR-26a,+miR-26b,+and+miR-29b+accelerate+osteogenic+differentiation+of+unrestricted+somatic+stem+cells+from+human+cord+blood.">MicroRNAs miR-26a, miR-26b, and miR-29b accelerate osteogenic differentiation of unrestricted somatic stem cells from human cord blood.</a> BMC Genomics. 14, 111 (2013).</li><li>Suga, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-formation+of+functional+adenohypophysis+in+three-dimensional+culture.">Self-formation of functional adenohypophysis in three-dimensional culture.</a> Nature. 480 (7375), 57-62 (2011).</li><li>Matsumoto, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Congenital+pituitary+hypoplasia+model+demonstrates+hypothalamic+OTX2+regulation+of+pituitary+progenitor+cells.">Congenital pituitary hypoplasia model demonstrates hypothalamic OTX2 regulation of pituitary progenitor cells.</a> Journal of Clinical Investigation. 130 (2), 641-654 (2019).</li><li>Kanie, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogenesis+of+anti-PIT-1+antibody+syndrome:+PIT-1+presentation+by+HLA+class+I+on+anterior+pituitary+cells.">Pathogenesis of anti-PIT-1 antibody syndrome: PIT-1 presentation by HLA class I on anterior pituitary cells.</a> Journal of the Endocrine Society. 3 (11), 1969-1978 (2019).</li><li>Lee, S. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+evolution+and+drug+response+in+patient-derived+organoid+models+of+bladder+cancer.">Tumor evolution and drug response in patient-derived organoid models of bladder cancer.</a> Cell. 173 (2), 515-528 (2018).</li><li>Yan, H. H. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comprehensive+human+gastric+cancer+organoid+biobank+captures+tumor+subtype+heterogeneity+and+enables+therapeutic+screening.">A comprehensive human gastric cancer organoid biobank captures tumor subtype heterogeneity and enables therapeutic screening.</a> Cell Stem Cell. 23 (6), 882-897 (2018).</li><li>Nolan, L. A., Kavanagh, E., Lightman, S. 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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...

Watch this Scientific Journal Video about Development of Organoids from Mouse Pituitary as In Vitro Model to Explore Pituitary Stem Cell Biology at JoVE.com

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, ...

Our organoid model is highly valuable to explore pituitary stem cell biology in homeostatic as well as pituitary remodeling conditions, such as in neonatal maturation of the gland, aging-associated functional decline, and tumor growth in the gland. To date, our pituitary organoid technique is the only available tool to reliably and robustly grow and expand primary mouse pituitary stem cells. Demonstrating the procedure will be Emma Laporte and Charlotte Nys, PhD students in my research group.To begin, wash the mouse head with deionized water to remove the blood. Spray 70%ethanol on the head to generate a sterile environment. Then, remove the skin between the ears using sterile surgical tools.To open the cranium, break the nose bridge with sterile scissors. Further open the cranium starting from the nose bridge toward the ears on both sides. Remove the cranium and the brain with sterile tweezers without touching the pituitary gland.Remove the diaphragma sellae with blunt tweezers. Then, under a stereomicroscope, separate the anterior lobe from the posterior and intermediate lobes. Carefully isolate the anterior lobe with blunt tweezers and collect it in a 10-milliliter Erlenmeyer flask containing three milliliters of medium A.Add two milliliters of pre-warmed 2.5%trypsin solution.Incubate at 37 degrees Celsius for 15 minutes. Add two milliliters of pre-warmed DNAse solution and swirl the Erlenmeyer flask 10 times. Let the pituitary sink to the bottom and remove the supernatant.Add two milliliters of pre-warmed trypsin inhibitor solution and incubate at 37 degrees Celsius for 10 minutes. Remove the supernatant when the pituitary sinks to the bottom. Add two milliliters of pre-warmed medium B and incubate for five minutes.Add two milliliters of pre-warmed medium C to this and incubate for 15 minutes. Let the pituitary sink to the bottom and remove the supernatant. Then, rinse it three times with pre-warmed medium C.Add two milliliters of pre-warmed medium C.Aspirate and expel the pituitary gland with a sterile, flame-polished Pasteur pipette multiple times until fragments are not visible anymore.Transfer the suspension to a 15-milliliter tube containing 4.5 milliliters of pre-warmed DNAse solution. Rinse the flask three times with two milliliters of pre-warmed medium C and transfer the suspension to the tube. Mix the collected cell suspension and filter it through a 40-micron cell strainer into a 30-milliliter tube.Rinse the tube and the cell strainer three times with two milliliters of medium C and transfer the suspension to the tube. Fill a glass Pasteur pipette with two milliliters of BSA. Position the tip of the pipette at the bottom of the tube and gently pipette out to form a visible density layer.Centrifuge at 190 times G for 10 minutes at four degrees Celsius. Remove the supernatant and resuspend the cell pellet in one milliliter of ice-cold advanced DMEM/F-12. Then, quantify the cells with a cell counter.Centrifuge the cell suspension at 190 times G for 10 minutes at four degrees Celsius and remove the supernatant. Resuspend the cell pellet in advanced DMEM/F-12 to reach a cell density of 1.1 times 10 to the 6th cells per milliliter. Add ECM to the desired volume of cell suspension at a ratio of 30:70 and mix well.Deposit a 30-microliter drop of this mixture in each well of a pre-warmed 48-well plate. Turn the plate upside down and let the ECM solidify at 37 degrees Celsius for 20 minutes. After the incubation, carefully add 250 microliters of pre-warmed pituitary organoid medium supplemented with 10-micromolar ROCK inhibitor.Every two to three days, change the medium without ROCK inhibitor for 10 to 14 days until the organoids are fully grown. To passage the organoids, first aspirate the medium gently and add 400 microliters of ice-cold advanced DMEM/F-12 to disintegrate the ECM. Then, collect the organoids in a microcentrifuge tube.Wash the well with 400 microliters of ice-cold advanced DMEM/F-12. Centrifuge the tube at 200 times G for five minutes at four degrees Celsius. Remove the supernatant and add 400 microliters of pre-warmed TrypLE Express enzyme.Mix by inverting the tube several times and incubate at 37 degrees Celsius for five minutes. Add 400 microliters of ice-cold advanced DMEM/F-12. Centrifuge at 200 times G for five minutes at four degrees Celsius and remove the supernatant.Resuspend the pellet with 100 microliters of ice-cold advanced DMEM/F-12. Narrow a P-200 tip and use the tip to dissociate the organoids by vigorously pipetting until organoid fragments are obtained. Add 800 microliters of advanced DMEM/F-12.Centrifuge at 190 times G for 10 minutes at four degrees Celsius and remove the supernatant. To passage the organoids, resuspend the pellet in an adequate volume of advanced DMEM/F-12 and add ECM at a ratio of 30:70. Mix well.Continue to seed and culture the organoids as described in the text manuscript. Dissociated single cells from the anterior lobe were seeded in ECM and grown in pituitary organoid medium. 14 days after seeding, the organoids were fully developed and reached a diameter of up to 500 micrometers.At this stage, the organoids exhibited cystic morphology with an epithelial layer enclosing a lumen. It is not recommended to use the wells in which dense structures appear after growth. For passaging, organoid fragments were used for seeding.Seven days after passaging, favorable organoid regrowth was observed with cystic structures. Dense organoids should be discarded, as the unfavorable organoids may take over the culture. Immunofluorescence staining analysis for the epithelial markers E-cadherin and cytokeratins 8 and 18 confirmed the epithelial character of the organoids.Expression of pituitary stem cell markers Sox2 and Trop2 demonstrated the stemness, and pituitary-specific marker LHX3 showed pituitary phenotype of the organoids. Expression of the marker Ki67 showed that the organoid-constituting cells are in a proliferative state. RTQ PCR analysis showed higher expression of stemness markers in the organoids than in the anterior lobe even after multiple passages, validating the enrichment of stem cells.It is important to plate the appropriate number of single cells in the ECM dome at the initial seeding, and when passaging the organoids, one must remember to re-seed fragments and not single cells.

development-organoids-from-mouse-pituitary-as-vitro-model-to-explore

Research

JoVE Journal

Developmental 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 ...