NH is funded by the MRC (MR/S009930/1) and the Wellcome Trust (204267/Z/16/Z), PD is funded by the MRC PhD DTP, KLF is funded by the BBSRC iCASE.

NOTE: All tissue culture work detailed below should be done in a Class II laminar flow hood.
1. Human induced pluripotent stem cell (hiPSCs) differentiation to the hindgut endoderm
<ol li="">
	<li>To coat a cell culture flask with extracellular matrix, first calculate the amount of the matrix needed. Here is an example of a 12-well plate being coated with Matrigel. Calculate the required amount of extracellular matrix to coat a 12 well plate. Use the following formula: 
	<img alt="Equation 1" src="/files/ftp_upload/61199/61199equ01.jpg" /> 
	NOTE: Coat cell culture dishes with extracellular matrix of choice. If using Matrigel, use a concentration of 0.035 mg/cm2 24 h prior to seeding for differentiation. Check batch concentration of extracellular matrix on product sheet and prepare smaller aliquots ahead of experiments according to manufacturer&#8217;s instructions.</li>
	<li>Dilute the required amount of extracellular matrix in cold DMEM media using a p1000 pipette.</li>
	<li>Mix well using a 5 mL stripette.</li>
	<li>Add 500&#956;L of diluted matrix to each well plate. Shake the plate gently to evenly distribute the diluted extracellular matrix and incubate for a minimum of 12 h at 37 &#176;C.</li>
	<li>Before seeding cells to the plate, wash each well with 500 &#181;L of PBS to remove excess extracellular matrix. This will prevent cell detachment during the differentiation.</li>
	<li>Leave the last wash in the wells until ready to seed to prevent the drying of extracellular matrix.</li>
	<li>To seed for hiPSCs differentiation towards definitive endoderm (DE) or hindgut (HG), take a flask of hiPSCs cultured in a maintenence media of choice (e.g.: essential 8 media) and aspirate the media. (Here, we seed cells at 25,000 cells/cm2). 
	NOTE: Seeding density is crucial for successful differentiation and needs to be optimized for each individual hiPSC cell line.</li>
	<li>Wash the cells in the flask with 5 mL of PBS. Aspirate the PBS.</li>
	<li>Add 2.5 mL of cell dissociation solution (e.g., TrypLE) and leave at RT for 4 min. Aspirate the cell dissociation solution and tap the flask gently to detach the cells.</li>
	<li>Wash the flask with 5 mL of DMEM media warmed to 37 &#176;C and collect all the cells into a 15 mL tube.</li>
	<li>Take 10 &#181;L of resuspended cell solution and measure the cell density with a hemocytometer.</li>
	<li>Take enough of the cell suspension to collect 1.05 x 106 cells and place in a 15 mL tube.</li>
	<li>Spin at 160 x g for 3 min. While the cells are being centrifuged, aspirate PBS from the extracellular matrix coated 12 well plate.</li>
	<li>Following centrifugation, aspirate the supernatant and resuspend the cell pellet in 12 mL of maintenance media with ROCK inhibitor (10 &#181;M).</li>
	<li>Using a p1000 pipette, add 1 mL of cell suspension to each well of the 12 well plate. Resuspend the cells well to ensure equal distribution between the wells.</li>
	<li>Shake the plate gently to distribute the cells within the wells of the plate but avoid swirling of the media as it will concentrate the cells in the middle of the wells.</li>
	<li>Place in a 37 &#176;C, 5% CO2 incubator.</li>
	<li>Change media to maintenance media (e.g., essential 8 media) only (no ROCK inhibitor) 24 h post seeding.</li>
	<li>To begin differentiation to DE, prepare endoderm basal media according to Table 1. 
	NOTE: At the start of the differentiation cells should be at between 60-80% confluence.</li>
	<li>Prepare 12 mL of DE media by adding Activin A (100 ng/mL) and Wnt3 (50 ng/mL) to the endoderm basal media.</li>
	<li>Warm up the media to 37 &#176;C.</li>
	<li>Aspirate media from each well of the tissue culture plate or flask.</li>
	<li>Start the DE differentiation by adding 1 mL of DE media to each well of the 12 well plate.</li>
	<li>At 24 h after starting DE differentiation (D1 DE), prepare fresh DE media and perform media change.</li>
	<li>Repeat step 1.24 at 48 h (D2 DE). If a lot of cell death is present at this stage, wash all wells with 500 &#181;L of PBS before media change. 
	NOTE: To confirm successful endoderm differentiation, perform flow cytometry to assess expression of SOX17. We normally expect &#62;80% of cells to be SOX17 positive by D3 DE. If expression of SOX17 is suboptimal cell seeding density should be optimized as well as concentrations of Act-A.</li>
	<li>Start HG differentiation at this point, i.e. 72 h post start of DE differentiation. Prepare 12 mL of HG media by adding CHIR99021 (3 &#181;M)&#160; and RA (1 &#956;M) to endoderm basal media (Table 1). 
	NOTE: At D3 DE the cell should have formed a uniform monolayer. Optimal DE differentiation is crucial for further stages of differentiation.</li>
	<li>Aspirate DE media.</li>
	<li>Start HG differentiation by adding 1 mL of HG media to each well of the 12 well plate.</li>
	<li>Continue the differentiation for 4 days with daily media changes. 
	NOTE: To determine success of DE posteriorization, perform flow cytometry to assess expression of CDX2. By D4 of HG differentiation we normally expect &#62;80% of cells to be CDX2 positive. If posteriorization is suboptimal, concentration of CHIR99021 should be optimized.</li>
	<li>To collect a sample for RNA extraction, aspirate differentiation media and wash the well with 500 ul of PBS.</li>
	<li>Aspirate PBS and add a suitable volume of cell lysis buffer from an RNA extraction kit.</li>
	<li>Using a p1000 pipette, scrape the bottom of the well to ensure lysis of all the cells.</li>
	<li>Aspirate the cell lysate and place in a clean tube.</li>
	<li>Proceed to RNA extraction or freeze the lysate at -20 &#176;C until ready for RNA extraction.</li>
	<li>For immunostaining, aspirate differentiation media and wash wells with 500 &#181;L of PBS.</li>
	<li>Aspirate the PBS and add 500 &#181;L of 4% PFA. 
	NOTE: PFA is toxic. Use appropriate PPE and follow local laboratory procedures for PFA disposal.</li>
	<li>Incubate at 4 &#176;C for 20 min.</li>
	<li>Remove PFA and wash the wells with 500 &#181;L of PBS three times.</li>
	<li>Leave the last PBS wash on the cells until ready to perform immunostaining.</li>
</ol>
2. Passaging of intestinal organoids
<ol>
	<li>Prepare intestinal basal media according to Table 2.</li>
	<li>To transfer from 2D to 3D cell culture, use a 6 well plate to generate DE cells form hiPSCS. Detach the monolayer of cells form the 6 cell plate using a 5 mL stripette.</li>
	<li>Collect cells into a 15 mL centrifuge tube, before centrifuging at 400 x g for 1 min to produce a cell pellet.</li>
	<li>Resuspend the cell pellet in intestinal growth media containing growth factors: SB202190 (10 &#181;M), A83-01 (500 nM), Gastrin (10 nM), Noggin (100 ng/&#181;L), EGF (500 ng/&#181;L), R-Spondin1 (100 ng/mL), CHIR99021 (6 &#181;M), ROCK inhibitor (10 &#181;M).</li>
	<li>Add appropriate volume of extracellular matrix dependent on the number of wells being plated into. This can be calculated using the equations below. 
	[1] Total volume of extracellular matrix/media (&#181;L) = 30 &#181;L * number of wells of 48-well plate 
	[2] Required volume of extracellular matrix (&#181;L) = Answer from [1] * 2/3 
	[3] Required volume of media (&#181;L) = Answer from [1] * 1/3</li>
	<li>Add 30 &#181;L of cell suspension to the center of each well of a 48-well plate.</li>
	<li>Incubate the plate in a 37&#160;&#176;C incubator for a minimum of 5 min to allow the extracellular matrix to set.</li>
	<li>Once the extracellular matrix domes have set, add 300 &#181;L of intestinal growth media containing all growth factors to each well.</li>
	<li>Return the plate to a 37&#160;&#176;C incubator with 5% CO2. 
	NOTE: If after 48 h no organoids can be observed and/or there is significant cell death ROCKi and NOGGIN concentrations may need to be optimized.</li>
	<li>To passage the organoids, observe the cells under a light microscope to assess density and size of organoids and determine whether they need passaging.</li>
	<li>Replace cell culture media with ice cold PBS. 
	NOTE: Organoid cultures will need splitting approximately every 5-7 days. Passaging of intestinal organoid cultures is required once there is an accumulation of cell debris evident within the organoid lumen and a yellowing of the surrounding intestinal growth medium.</li>
	<li>Mechanically detach organoids and extracellular matrix sphere from the plate using a scraping motion with a 5 mL stripette.</li>
	<li>Collect the organoids from each well into a 15 mL centrifuge tube.</li>
	<li>Centrifuge organoid suspension at 400 x g for 1 min to pellet the organoids. Aspirate the supernatant until reaching the top of the cell pellet, being careful when having reached the visible extracellular matrix layer.</li>
	<li>Resuspend the pellet in 15 mL of ice cold PBS. 
	NOTE: This step serves to wash any remaining extracellular matrix that was not removed in the initial spin.</li>
	<li>Centrifuge at 400 x g for 1 min. Aspirate the media until the pellet, containing the organoids.</li>
	<li>Resuspend in 1 mL of ice cold PBS. Using a p200 pipette, manually disrupt the intact organoids by pipetting up and down several times. 
	NOTE: Observe the size of the organoids using light microscope to determine whether they need to be further dissociated.</li>
	<li>Add 9 mL of media without growth factors.</li>
	<li>Centrifuge at 400 x g for 1 min. Aspirate the supernatant until the organoids pellet.</li>
	<li>Calculate the required amount of extracellular matrix and media using the equations below: 
	[1] Total volume of extracellular matrix/media (&#181;L) = 30 &#181;L * number of wells of 48-well plate 
	[2] Required volume of extracellular matrix (&#181;L) = Answer from [1] * 2/3 
	[3] Required volume of media (&#181;L) = Answer from [1] * 1/3</li>
	<li>Resuspend the organoid pellet in the calculated volume of intestinal media with growth factors (including Noggin &#38; ROCK inhibitor).</li>
	<li>Add the required volume of extracellular matrix into this cell suspension and resuspend to ensure even distribution of organoids.</li>
	<li>Pipette 30 &#181;L of this suspension into the center of each well of a 48 well plate (preferably pre-heated in a 37 &#176;C incubator).</li>
	<li>Return the plate to a 37 &#176;C incubator for 5 min until the extracellular matrix has set.</li>
	<li>Prepare intestinal media with growth factors (+ ROCK inhibitor) (approximately 17 mL per 48 well plate).</li>
	<li>Add 300 &#181;L to each well.</li>
	<li>Incubate at 37 &#176;C at 5% CO2.</li>
	<li>Following passaging, aspirate the media for the intestinal organoids and replace with fresh intestinal media with growth factors (without Noggin &#38; ROCK inhibitor) every 2-4 days. 
	NOTE:&#160;If after 48 hours no organoids can be observed and/or there is significant cell death ROCKi and NOGGIN concentrations may need to be optimized. 
	NOTE: Intestinal organoids respond to a range of inflammatory mediators in vivo. TNF&#945; is a cell signaling protein involved in several inflammatory processes.</li>
	<li>To trigger an inflammatory response in intestinal organoids, prepare TNF&#945; at a concentration of 40 ng/mL in basal media only.</li>
	<li>Aspirate media from 48 well plate.</li>
	<li>Add 300 &#181;L of prepared basal media containing 40 ng/mL TNF&#945;.</li>
	<li>Incubate plate at 37&#160;&#176;C for 48 hours to replicate a pro-inflammatory environment. 
	NOTE: Intestinal organoids respond to a range of inflammatory mediators in vivo. TNF&#945; is a cell signaling protein involved in several inflammatory processes.</li>
	<li>Dispose of any remaining cells by aspiration into a vacuum trap containing 5% Trigene.</li>
</ol>

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The authors have nothing to disclose.

Here, we describe a protocol for the differentiation of human pluripotent cells into human intestinal organoids. We demonstrate their use to study inflammation; however, this can be applied to various contexts, and coupled with CRISPR/Cas9 gene editing approaches14, on any genetic background. Once differentiated, following the natural developmental differentiation sequence of definitive endoderm, hindgut endoderm and then intestinal epithelium, the resulting organoids can be continually cultured and passaged for more than 12 months.
A critical aspect of this protocol is the initial plating density of undifferentiated stem cells prior to endoderm differentiation. If this is not optimized sufficiently cells will likely die during the initial DE differentiation step (if cells are too sparse) or reduce the efficiency of the DE differentiation (if the cells are too dense). The correct starting density needs to be optimized for the cell line being used, and the correct density should generate a monolayer by the end of DE D3. Flow cytometry should be used to determine efficiency of DE specification and we typically see &#62;80% of cells positive for SOX17 and/or CXCR4. When the number of SOX17 positive cells is less than 60%, the efficiency of HG patterning is impacted, which results in fewer organoids forming when transferred to the extracellular matrix. This will ultimately cause the resulting organoid cultures to fail. To determine if patterning DE to HG has been successful, we assess the number of CDX2 positive cells by flow cytometry and typically expect to see &#62;80% positive cells. Again, if the number of CDX2 positive cells falls below 50% this will have an adverse effect on the number of intestinal organoids that are generated when transferred to 3D extracellular matrix culture.
Following transfer of 2D monolayers into 3D culture, small compact spheres should appear 24-48 h following transfer. Large sheets of dead cells can appear depending on the differentiation efficiency for the cell line used. Instead of immediately passaging cultures to remove these debris, we allow the organoids to fully form and develop their more complex, folded structure. Waiting 7-10 days before attempting the first passing ensures that sufficient dividing cells are present to generate many new intestinal organoids. Any debris that are still present in the culture can be easily removed during the passaging process by slowly spinning the dissociated organoid/debris mixtures in a tube with sufficient speed to pellet the organoids but leave sheet of cells floating in the media. Media and cellular debris can then be aspirated so that only the pellet of organoids remains.
The limitation of this approach is that hiPSC-derived cell types are not often fully mature in terms of gene expression and functional profiles. To determine if hiPSC-derived intestinal tissue is suitable for specific applications the organoids should be characterized for different cell types including enterocytes (VIL), enteroendocrine cells (neurog3), goblet cells (MUC2), transient amplifying cells (CD133), paneth cells (FZD5) and LGR5+ stem cells (LGR5) to determine the organoid cellular composition.
Overall, the major advantage of this protocol over many other organoid differentiation protocols is that this culture platform is very cost-effective due to replacement of several recombinant proteins and conditioned media preparations with small molecules15,16. The differentiation into HG is very simple and fast and can be applied to both human embryonic and induced pluripotent stem cells with identical outcomes. When followed rigorously, and optimized for the cell lines being used it provides a relatively simple model platform free of contaminating mesenchymal cells that can then be applied to study intestinal epithelium in a variety of context including inflammation, host pathogen interactions7. Intestinal fibrosis modeling could be investigated by providing pro-fibrotic stimuli and then assessing expression of extracellular matrix proteins such as collagen, laminin and fibronectin by QPCR, western blot and ELISA. Using CRISPR/Cas9 gene editing techniques on undifferentiated stem cell lines prior to differentiation allows for the creation of gene knockout or protein overexpression organoids that could be used to create disease specific organoids and more complex disease models14,17,18.

The properties of pluripotent stem cells (PSCs), such as self-renewal and the ability to differentiate to any cell type of the human body, make them valuable tools in the study of development, disease pathology and drug testing1. Human induced pluripotent stem cells (hiPSC) are particularly useful for disease modelling studies as they can be derived from patients, directly capturing the genome responsible for the disease phenotype2,3. Such hiPSCs can be differentiated to the cell type affected by the genetic defect allowing careful examination of molecular mechanism of the disease4.
Differentiation protocols for human PSCs aim to direct differentiation of cells via the main developmental stages by activation or inhibition of specific signaling pathways governing lineage commitment and specification. Maintenance of hiPSC in a pluripotent state requires moderate levels of Activin A (Act-A) signaling, while a high dosage of Act-A for 3 days commits hiPSC to a definitive endoderm (DE) fate5,6. Act-A and Wnt pathways direct anterior-posterior identity of the DE. Signaling by Act-A induces foregut (FG) markers such as HHEX, HNF4&#945; and GATA4, while blocking expression of hindgut (HG) genes such as CDX2. Wnt signaling induces posteriorization of the DE, which then adopts a HG gene expression profile7,8. Once HG cell identity is established, differentiation can be moved from 2D to 3D and directed towards the formation of intestinal organoids.
Intestinal organoids are typically cultured in a 3D extracellular matrix (e.g., Matrigel or other compatible ECMs) based culture system9, comprised of laminin, collagen IV and entactin and enriched with growth factors such as EGF, FGF, PDGF and IGF-1 to contribute to the support survival and proliferation. The organoids are cultured in a defined medium containing Gastrin, Noggin and CHIR to stimulate and support intestinal stem cell growth and proliferation during long-term culture.
After intestinal epithelial cells are embedded into the extracellular matrix, intestinal crypts begin to form and expand eventually forming spheroids. These mature into organoid structures that mimic physiological functioning of intestinal epithelium. Organoids can typically be cultured for more than 1 year without significant loss of functional and gene expression profiles. Passaging is required on a weekly basis using enzymatic digestion of the organoids into smaller fragments which then self-reassemble into complete organoids.
Established organoid lines can be used as a reliable model of numerous disorders linked to the intestine, including Crohn&#8217;s Disease, Ulcerative Colitis and colorectal cancers10,11,12,13. This is a preferred model to animal cells as they express human genes linked to these disorders and respond to external stimuli more closely resembling that which occurs in human tissue in vivo.

<table><tbody><tr><td>A83-01</td><td>Tocris</td><td>2939</td><td></td></tr><tr><td>Activin A</td><td>R&amp;amp;D</td><td>338-AC</td><td></td></tr><tr><td>Advanced DMEM/F12 (1X)</td><td>Life Technologies</td><td>12654-010</td><td></td></tr><tr><td>B27 supplement</td><td>Gibco</td><td>17504044</td><td></td></tr><tr><td>CHIR99021</td><td>Sigma</td><td>SML1046-5MG</td><td></td></tr><tr><td>Epidermal Growth Factor</td><td>R&amp;amp;D Systems</td><td>236-EG-01M</td><td></td></tr><tr><td>Gastrin</td><td>Sigma Aldrich</td><td>G9145</td><td></td></tr><tr><td>GlutaMAX (100X)</td><td>Life Technologies</td><td>15630-056</td><td></td></tr><tr><td>Growth Factor reduced Matrigel</td><td>BD</td><td></td><td></td></tr><tr><td>HEPES Buffer solution (1M)</td><td>Life Technologies</td><td>15630-080</td><td></td></tr><tr><td>N2 Supplement (100X)</td><td>Gibco</td><td>17502-048</td><td></td></tr><tr><td>N-acetyl-cysteine</td><td>Sigma Aldrich</td><td>A7250</td><td></td></tr><tr><td>Nicotinamide</td><td>Sigma Aldrich</td><td>N0636</td><td></td></tr><tr><td>Noggin</td><td>R&amp;amp;D Systems</td><td>6057-NG</td><td></td></tr><tr><td>Non-essential amino acids</td><td>Gibco</td><td>11140-050</td><td></td></tr><tr><td>Paraformaldehyde</td><td>VWR</td><td>9713.5</td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>Gibco</td><td>15140122</td><td></td></tr><tr><td>Phosphate Buffered Saline</td><td>Gibco</td><td>14190-094</td><td></td></tr><tr><td>Retinoic Acid</td><td>Sigma</td><td>302-79-4</td><td></td></tr><tr><td>ROCK inhibitor</td><td>Tocris</td><td>1254/1</td><td></td></tr><tr><td>ROCK inhibitor Y-27632</td><td>Tocris</td><td>1254</td><td></td></tr><tr><td>RPMI</td><td>Sigma</td><td>R8758-500ml</td><td></td></tr><tr><td>R-Spondin-1</td><td>Peprotech</td><td>120-38</td><td></td></tr><tr><td>SB202190</td><td>Tocris</td><td>1264</td><td></td></tr><tr><td>TrypLe Express</td><td>Gibco</td><td>12604-021</td><td></td></tr><tr><td>Wnt 3a</td><td>R&amp;amp;D</td><td>5036-WN</td><td></td></tr></tbody></table>

generation of hipsc-derived intestinal organoids for developmental and disease modelling applications

hiPSC-derived intestinal organoids are epithelial structures that self-assemble from differentiated cells into complex 3D structures, representative of the human intestinal epithelium, in which they exhibit crypt/villus-like structures. Here, we describe the generation of hiPSC-derived intestinal organoids by the stepwise differentiation of hiPSCs into definitive endoderm, which is then posteriorized to form hindgut epithelium before being transferred into 3D culture conditions. The 3D culture environment consists of extracellular matrix (ECM) (e.g., Matrigel or other compatible ECM) supplemented with SB202190, A83-01, Gastrin, Noggin, EGF, R-spondin-1 and CHIR99021. Organoids undergo passaging every 7 days, where they are mechanically disrupted before transfer to fresh extracellular matrix and allowed to expand. QPCR and immunocytochemistry confirm that hiPSC-derived intestinal organoids contain mature intestinal epithelial cell types including goblet cells, Paneth cells and enterocytes. Additionally, organoids show evidence of polarization by expression of villin localized on the apical surface of epithelial cells.
The resulting organoids can be used to model human intestinal development as well as numerous human intestinal diseases including inflammatory bowel disease. To model intestinal inflammation, organoids can be exposed to inflammatory mediators such as TNF-&#945;, TGF-&#946;, and bacterial LPS. Organoids exposed to proinflammatory cytokines display an inflammatory and fibrotic phenotype in response. Pairing of healthy versus hiPSCs derived from patients with IBD may be useful in understanding mechanisms driving IBD. This may reveal novel therapeutic targets and novel biomarkers to assist in early disease diagnosis.

A schematic of the differentiation protocol is shown in Figure 1A. On day 1 of the protocol, hiPSCs should be compact and forming small colonies with total confluence approximately 50-60%. 24 hours following induction of differentiation towards DE cells begin to migrate away from the stem cell colonies to form a monolayer of cells. This continues over the following 3 days and should form a complete monolayer on D3 of DE differentiation (Figure 1). Gene expression should be monitored over the course of differentiation with pluripotency markers (OCT4, NANOG, SOX2) highly expressed on D0 and rapidly downregulated during DE differentiation. During DE differentiation T expression should peak on D1, followed by EOMES and MIXL on D2. On D2 DE genes (SOX17, FOXA2, GATA4, CXCR4) should begin to be expressed and peak at D3 (Figure 2 and Figure 3). Cells should be a monolayer by DE D3 and can then be posteriorized into hindgut endoderm. During the posteriorization event 3D structures will start to form as early as D2. However, sometimes they will only start to appear at D4 or not at all; this is not always indicative of whether or not the cells will proceed and form intestinal organoids (Figure 1). During HG specification CDX2 and HNF4a expression should be induced and increase over time (Figure 3).
Following transfer of 2D sheets of cells into extracellular matrix, clumps of 2D cells will be observed for the first 24 h. After 48 h sheets of cells should begin to auto organize into more compacted 3D spheroid structures that are initially small (Figure 4A) then gradually increase in size and complexity over 7-10 days of culture (Figure 4B &#38; Figure 4C). Organoids should not be passaged until they have achieved a clear organoid/spheroid morphology with obvious epithelium with the lumen facing towards the center of the organoid/spheroid (Figure 4D). At this stage immunocytochemistry can be used to confirm expression of intestinal markers such as villin and CDX2 (Figure 5). Not all clumps of 2D cells will develop into organoids and there will be some contaminating dead cells within the extracellular matrix. These dead sheets of cells should be ignored until the surviving cells have formed large organoids and are ready for passaging.
To model inflammation, TNF&#945; can be added to the tissue culture medium for 24-48 h. Following incubation with proinflammatory molecules, organoids are harvested using the same technique used for their isolation and passaging and then lysed using a cell buffer compatible application such as QPCR or western blotting. If shorter exposures are required, organoids should first be removed from the extracellular matrix and exposed to TNF&#945; in suspension using a 1.5 mL tube. Treatment of intestinal organoids with TNF&#945; for 48 h typically induces expression of pro-inflammatory markers (TNF&#945;, IL1B, IL8, IL23) while negatively affecting expression of intestinal epithelial markers (LGR5, VIL) (Figure 6).
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="2">Endoderm basal media</td>
			<td>50 mL</td>
		</tr>
		<tr>
			<td colspan="2">RPMI 1640</td>
			<td>48.5 mL</td>
		</tr>
		<tr>
			<td colspan="2">B27 Supplement</td>
			<td>1 mL</td>
		</tr>
		<tr>
			<td colspan="2">1% NEAA</td>
			<td>0.5 mL</td>
		</tr>
	</tbody>
</table>
Table 1: Composition of endodermal basal media for endoderm differentiation.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="2">Intestinal Basal Media</td>
			<td>50 mL</td>
		</tr>
		<tr>
			<td colspan="2">Advanced DMEM/F12</td>
			<td>46.5 mL</td>
		</tr>
		<tr>
			<td colspan="2">HEPES Buffer</td>
			<td>0.5 mL</td>
		</tr>
		<tr>
			<td colspan="2">GlutaMAX</td>
			<td>0.5 mL</td>
		</tr>
		<tr>
			<td colspan="2">Nicotinamide</td>
			<td>0.5 mL</td>
		</tr>
		<tr>
			<td colspan="2">N2 Supplement</td>
			<td>0.5 mL</td>
		</tr>
		<tr>
			<td colspan="2">B27 Supplement</td>
			<td>1.0 mL</td>
		</tr>
		<tr>
			<td colspan="2">Pen/Strep</td>
			<td>0.5 mL</td>
		</tr>
	</tbody>
</table>
Table 2: Composition of intestinal basal media for culture of intestinal organoids
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61199/61199fig01.jpg" /> 
Figure 1: Morphological changes during differentiation of hiPSC via definitive endoderm to the hindgut lineage. 
(A)&#160;Schematic overview of intestinal differentiation protocol. This cell line of hiPSC forms loose colonies of small cells with a high nucleus to cytoplasm ratio. As the differentiation proceeds, the cells undergo changes consistent with transition from the epithelial to the mesenchymal phenotype and by DE D3 form a uniform monolayer. Once appropriate signals are delivered, DE cells elongate and form a more densely packed monolayer with 3D spheroids appearing as soon as HD D3 but this is dependent upon cell line used and is not a requirement for transition to 3D culture (B). Scale bar: 100 &#181;m. <a href="https://www.jove.com/files/ftp_upload/61199/61199fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61199/61199fig02.jpg" /> 
Figure 2: Differentiation of hiPSCs to HG endoderm induces expression of endodermal genes. 
Immunostaining of hiPSC differentiating to definitive endoderm shows changes in expression of TFs at protein level. Pluripotency markers (NANOG and OCT4) are downregulated by DE D3 (A). Expression of mesendoderm marker BRA(T) is present at D1 of the protocol (B) and DE specific TFs SOX17 and FOXA2 appear on D2 (B &#38; C). Scale bar: 200 mm. <a href="https://www.jove.com/files/ftp_upload/61199/61199fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61199/61199fig03v2.jpg" /> 
Figure 3: Gene expression changes by qPCR during hiPSC differentiation to hindgut endoderm (HG). 
Genes associated with pluripotency are downregulated (OCT4, NANOG, SOX2) followed by transient expression of mesendoderm genes (T, EOMES, MIXL1), and finally expression of DE genes (SOX17, FOXA2, CXCR4) and hindgut genes (CDX2, GATA4, HNF4a). Data presented as mean &#177;SD.&#160;<a href="https://www.jove.com/files/ftp_upload/61199/61199fig03largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61199/61199fig04.jpg" /> 
Figure 4: HG endoderm self-assembles to form 3D intestinal organoids in 3D extracellular matrix culture. 
HG endoderm is transferred into a suitable 3D extracellular matrix culture and initially forms small solid clumps of cells (A). Clumps of HG endoderm expand over 7-10 days of culture (B) and then become asymmetrical and begin to form a more complex epithelium (C) eventually giving rise to organoids with clear epithelial morphology and a luminal surface facing towards the centre of the organoid (D). Scale bars = 50 &#181;m <a href="https://www.jove.com/files/ftp_upload/61199/61199fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/61199/61199fig05.jpg" /> 
Figure 5: Established hiPSC-derived intestinal organoids express intestinal markers. 
Immunocytochemistry showing expression of CDX2 and Villin. Scale bars = 100 &#181;m <a href="https://www.jove.com/files/ftp_upload/61199/61199fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/61199/61199fig06v2.jpg" /> 
Figure 6: Effect of TNF&#945; on the inflammatory profile &#38; intestinal cell expression of healthy intestinal organoids. 
Inflammatory profile of healthy colonic organoids following 48 hour treatment with TNF&#945; (40 ng/mL). Expression of pro-inflammatory markers (TNF&#945;, IL-8 &#38; IL-23) increases following exposure to TNF&#945;, while at the same time expression of intestinal epithelial markers (LGR5, VIL) are downregulated. Statistical analyses were performed by two-sided student&#8217;s t-test. Data are expressed as mean &#177; SD of each group. *P &#60; .01; **P &#60; .001; ***P &#60; .0001. (n=3). <a href="https://www.jove.com/files/ftp_upload/61199/61199fig06largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Generation of hiPSC-Derived Intestinal Organoids for Developmental and Disease Modelling Applications at JoVE.com

Generation of hiPSC-Derived Intestinal Organoids for Developmental and Disease Modelling Applications

This protocol allows for the differentiation of human pluripotent cells into intestinal organoids. The protocol mimics normal human development by differentiating cells into a population of definitive endoderm, hindgut endoderm and then intestinal epithelium. This makes the protocol suitable for studying both intestinal development as well as disease modelling applications.

hiPSC-derived intestinal organoids are epithelial structures that self-assemble from differentiated cells into complex 3D structures, representative of ...

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Biology

2.5K Views.  University of Nottingham. Deze methode biedt stapsgewijze instructies voor het genereren van door de mens geïnduceerde pluripotente stamcel-afgeleide darmorganoïden door differentiatie in definitief endoderm, vervolgens in het epitheel van de dikke darm en ten slotte in de overdracht naar 3D-kweekomstandigheden. Resultaten verkregen uit door de mens geïnduceerde pluripotente stamcel-afgeleide darmorganoïden tonen een grotere vertaalbaarheid aan dan gegevens van eencellige culturen, terwijl ze ook praktischer zijn ...

Video: Generation of hiPSC-Derived Intestinal Organoids for Developmental and Disease Modelling Applications

Real-time Measurement of Epithelial Barrier Permeability in Human Intestinal Organoids

Advances in 3D culture of intestinal tissues obtained through biopsy or generated from pluripotent stem cells via directed differentiation, have resulted in sophisticated in vitro models of the intestinal mucosa. Leveraging these emerging model systems will require adaptation of tools and techniques developed for 2D culture systems and animals. Here, we describe a technique for measuring epithelial barrier permeability in human intestinal organoids in real-time. This is accomplished by microinjection of fluorescently-labeled dextran and imaging on an inverted microscope fitted with epifluorescent filters. Real-time measurement of the barrier permeability in intestinal organoids facilitates the generation of high-resolution temporal data in human intestinal epithelial tissue, although this technique can also be applied to fixed timepoint imaging approaches. This protocol is readily adaptable for the measurement of epithelial barrier permeability following exposure to pharmacologic agents, bacterial products or toxins, or live microorganisms. With minor modifications, this protocol can also serve as a general primer on microinjection of intestinal organoids and users may choose to supplement this protocol with additional or alternative downstream applications following microinjection.

This protocol describes the measurement of epithelial barrier permeability in real-time following pharmacologic treatment in human intestinal organoids using fluorescent microscopy and live cell microscopy.

Advances in 3D culture of intestinal tissues obtained through biopsy or generated from pluripotent stem cells via directed differentiation, have resulted ...

Developmental Biology

Generation, Maintenance, and Characterization of Human Pluripotent Stem Cell-derived Intestinal and Colonic Organoids

Intestinal regional specification describes a process through which unique morphology and function are imparted to defined areas of the developing gastrointestinal (GI) tract. Regional specification in the intestine is driven by multiple developmental pathways, including the bone morphogenetic protein (BMP) pathway. Based on normal regional specification, a method to generate human colonic organoids (HCOs) from human pluripotent stem cells (hPSCs), which include human embryonic stem cells (hES) and induced pluripotent stem cells (iPSCs), was developed. A three-day induction of BMP signaling sufficiently patterns mid/hindgut tube cultures into special AT-rich sequence-binding protein 2 (SATB2)-expressing HCOs containing all of the main epithelial cell types present in human colon as well as co-developing mesenchymal cells. Omission of BMP (or addition of the BMP inhibitor NOGGIN) during this critical patterning period resulted in the formation of human intestinal organoids (HIOs). HIOs and HCOs morphologically and molecularly resemble human developing small intestine and colon, respectively. Despite the utility of HIOs and HCOs for studying human intestinal development, the generation of HIOs and HCOs is challenging. This paper presents methods for generating, maintaining, and characterizing HIOs and HCOs. In addition, the critical steps in the protocol and troubleshooting recommendations are provided.

Here, detailed methods for generating, maintaining, and characterizing human pluripotent stem cell-derived small intestinal and colonic organoids are described. These methods are designed to improve reproducibility, expand scalability, and decrease the working time required for plating and passaging of organoids.

Intestinal regional specification describes a process through which unique morphology and function are imparted to defined areas of the developing ...

Innervation of Human Intestinal Organoids

The complexity of intestinal cytoarchitecture and function poses significant challenges for the creation of the bioengineered small intestine. Techniques for generating human intestinal organoids (HIOs) resembling human small intestine have been previously reported. HIOs contain epithelium and mesenchyme but lack other critical components of functional intestine such as the enteric nervous system (ENS), immune cells, vasculature, and microbiome. Two independent research groups have published distinct methods to innervate HIOs with an ENS. Here we discuss a unique method of incorporating the ENS into an HIO-derived bioengineered small intestine, which utilizes components of these prior reports to optimize progenitor cell identity as well as developmental timing.
Human pluripotent stem cells (hPSCs) are differentiated to independently generate HIOs and enteric neural crest cells (ENCCs) by temporal regulation of differentiation markers over a period of several days per published protocols. Once HIOs reach the mid-hindgut spheroid stage (approximately day 8), day 15-21 ENCC spheroids are dissociated, co-cultured with HIOs, and suspended within clear three-dimensional (3D) basement membrane matrix droplets. HIO + ENCC co-cultures are maintained in vitro for 28-40 days before transplantation into &#62;9-week-old immunodeficient mice for further development and maturation. Transplanted HIOs (tHIOs) with ENS can be harvested 4-20 weeks later. This method integrates elements from two previously published techniques by utilizing ENCCs generated from hPSCs and co-culturing them with HIOs at an early stage of development to maximize exposure to early developmental cues that likely contribute to the formation of a more mature intestinal morphology.

Human intestinal organoids must be innervated to better recapitulate the structure and function of the native human intestine. Here, we present one method for incorporating an enteric nervous system into these constructs.

The complexity of intestinal cytoarchitecture and function poses significant challenges for the creation of the bioengineered small intestine. Techniques ...

Using Human Induced Pluripotent Stem Cell-derived Intestinal Organoids to Study and Modify Epithelial Cell Protection Against Salmonella and Other Pathogens

The intestinal &#8216;organoid&#8217; (iHO) system, wherein 3-D structures representative of the epithelial lining of the human gut can be produced from human induced pluripotent stem cells (hiPSCs) and maintained in culture, provides an exciting opportunity to facilitate the modeling of the epithelial response to enteric infections. In vivo, intestinal epithelial cells (IECs) play a key role in regulating intestinal homeostasis and may directly inhibit pathogens, although the mechanisms by which this occurs are not fully elucidated. The cytokine interleukin-22 (IL-22) has been shown to play a role in the maintenance and defense of the gut epithelial barrier, including inducing a release of antimicrobial peptides and chemokines in response to infection.
We describe the differentiation of healthy control hiPSCs into iHOs via the addition of specific cytokine combinations to their culture medium before embedding them into a basement membrane matrix-based prointestinal culture system. Once embedded, the iHOs are grown in media supplemented with Noggin, R-spondin-1, epidermal growth factor (EGF), CHIR99021, prostaglandin E2, and Y-27632 dihydrochloride monohydrate. Weekly passages by manual disruption of the iHO ultrastructure lead to the formation of budded iHOs, with some exhibiting a crypt/villus structure. All iHOs demonstrate a differentiated epithelium consisting of goblet cells, enteroendocrine cells, Paneth cells, and polarized enterocytes, which can be confirmed via immunostaining for specific markers of each cell subset, transmission electron microscopy (TEM), and quantitative PCR (qPCR). To model infection, Salmonella enterica serovar Typhimurium SL1344 are microinjected into the lumen of the iHOs and incubated for 90 min at 37 &#176;C, and a modified gentamicin protection assay is performed to identify the levels of intracellular bacterial invasion. Some iHOs are also pretreated with recombinant human IL-22 (rhIL-22) prior to infection to establish whether this cytokine is protective against Salmonella infection.

Using Human Induced Pluripotent Stem Cell-derived Intestinal Organoids to Study and Modify Epithelial Cell Protection Against Salmonella and Other Pathogens

Human induced pluripotent stem cell (hiPSC)-derived intestinal organoids offer exciting opportunities to model enteric diseases in vitro. We demonstrate the differentiation of hiPSCs into intestinal organoids (iHOs), the stimulation of these iHOs with cytokines, and the microinjection of Salmonella Typhimurium into the iHO lumen, enabling the study of an epithelial invasion by this pathogen.

The intestinal &#8216;organoid&#8217; (iHO) system, wherein 3-D structures representative of the epithelial lining of the human gut can be produced from ...

Immunology and Infection

Combining Human Organoids and Organ-on-a-Chip Technology to Model Intestinal Region-Specific Functionality

The intestinal mucosa is a complex physical and biochemical barrier that fulfills a myriad of important functions. It enables the transport, absorption, and metabolism of nutrients and xenobiotics while facilitating a symbiotic relationship with microbiota and restricting the invasion of microorganisms. Functional interaction between various cell types and their physical and biochemical environment is vital to establish and maintain intestinal tissue homeostasis. Modeling these complex interactions and integrated intestinal physiology in vitro is a formidable goal with the potential to transform the way new therapeutic targets and drug candidates are discovered and developed.
Organoids and Organ-on-a-Chip technologies have recently been combined to generate human-relevant intestine chips suitable for studying the functional aspects of intestinal physiology and pathophysiology in vitro. Organoids derived from the biopsies of the small (duodenum) and large intestine are seeded into the top compartment of an organ chip and then successfully expand&#160;as monolayers while preserving the distinct cellular, molecular, and functional features of each intestinal region. Human intestine tissue-specific microvascular endothelial cells are incorporated in the bottom compartment of the organ chip to recreate the epithelial-endothelial interface. This novel platform facilitates luminal exposure to nutrients, drugs, and microorganisms, enabling studies of intestinal transport, permeability, and host-microbe interactions.
Here, a detailed protocol is provided for the establishment of intestine chips representing the human duodenum (duodenum chip) and colon (colon chip), and their subsequent culture under continuous flow and peristalsis-like deformations. We demonstrate methods for assessing drug metabolism and CYP3A4 induction in duodenum chip using prototypical inducers and substrates. Lastly, we provide a step-by-step procedure&#160;for the in vitro modeling of interferon gamma (IFN&#947;)-mediated barrier disruption (leaky gut syndrome) in a colon chip, including methods for evaluating the alteration of paracellular permeability, changes in cytokine secretion, and transcriptomic profiling of the cells within the chip.

Biopsy-derived intestinal organoids and organ-on-a-chips technologies are combined into a microphysiological platform to recapitulate region-specific intestinal functionality.

The intestinal mucosa is a complex physical and biochemical barrier that fulfills a myriad of important functions. It enables the transport, absorption, ...

Bioengineering

hiPSC-Derived Vascular Organoids for Drug Screening and Disease Modeling

Vascular Organoid Generation from Human-Induced Pluripotent Stem Cells

Vascular organoids derived from human induced pluripotent stem cells (hiPSCs) recapitulate the cell type diversity and complex architecture of human vascular networks. This three-dimensional (3D) model holds substantial potential for vascular pathology modeling and in vitro drug screening. Despite recent advances, a key technical challenge remains in reproducibly generating organoids with consistent quality, which is crucial for downstream assays and applications. Here, a modified protocol is presented that improves both the homogeneity and reproducibility of vascular organoid generation. The modified protocol incorporates the use of microwells and the CEPT cocktail (chroman 1, emricasan, polyamines, and the integrated stress response inhibitor, trans-ISRIB) to improve embryoid body formation and cell survival. Differentiated, mature vascular organoids generated using this protocol are characterized by whole-mount 3D immunofluorescence microscopy to analyze their morphology and complex vasculature. This protocol enables the production of high-quality vascular organoids in a scalable manner, potentially facilitating their use in disease modeling and drug screening applications.

Author Spotlight: Improving Reproducibility in Vascular Organoids Using ROCK Inhibitors and Microwell Confinement

This protocol presents a method for generating vascular organoids using human pluripotent stem cells.

Vascular organoids derived from human induced pluripotent stem cells (hiPSCs) recapitulate the cell type diversity and complex architecture of human ...

Organoid-Derived Epithelial Monolayer: A Clinically Relevant In Vitro Model for Intestinal Barrier Function

In the past, intestinal epithelial model systems were limited to transformed cell lines and primary tissue. These model systems have inherent limitations as the former do not faithfully represent original tissue physiology, and the availability of the latter is limited. Hence, their application hampers fundamental and drug development research. Adult stem-cell-based organoids (henceforth referred to as organoids) are miniatures of normal or diseased epithelial tissue from which they are derived. They can be established very efficiently from different gastrointestinal (GI) tract regions, have long-term expandability, and simulate tissue- and patient-specific responses to treatments in vitro. Here, the establishment of intestinal organoid-derived epithelial monolayers has been demonstrated along with methods to measure epithelial barrier integrity, permeability and transport,&#160;antimicrobial protein secretion, as well as histology. Moreover, intestinal organoid-derived monolayers can be enriched with proliferating stem and transit-amplifying cells as well as with key differentiated epithelial cells. Therefore, they represent a model system that can be tailored to study the effects of compounds on target cells and their mode of action. Although organoid cultures are technically more demanding than cell lines, once established, they can reduce failures in the later stages of drug development as they truly represent in vivo epithelium complexity and interpatient heterogeneity.

Here, we describe the preparation of human organoid-derived intestinal epithelial monolayers for studying intestinal barrier function, permeability, and transport. As organoids represent original epithelial tissue response to external stimuli, these models combine the advantages of expandability of cell lines and the relevance and complexity of primary tissue.

In the past, intestinal epithelial model systems were limited to transformed cell lines and primary tissue. These model systems have inherent limitations ...

Culture Methods to Study Apical-Specific Interactions using Intestinal Organoid Models

The lining of the gut epithelium is made up of a simple layer of specialized epithelial cells that expose their apical side to the lumen and respond to external cues. Recent optimization of in vitro culture conditions allows for the re-creation of the intestinal stem cell niche and the development of advanced 3-dimensional (3D) culture systems that recapitulate the cell composition and the organization of the epithelium. Intestinal organoids embedded in an extracellular matrix (ECM) can be maintained for long-term and self-organize to generate a well-defined, polarized epithelium that encompasses an internal lumen and an external exposed basal side. This restrictive nature of the intestinal organoids presents challenges in accessing the apical surface of the epithelium in vitro and limits the investigation of biological mechanisms such as nutrient uptake and host-microbiota/host-pathogen interactions. Here, we describe two methods that facilitate access to the apical side of the organoid epithelium and support the differentiation of specific intestinal cell types. First, we show how ECM removal induces an inversion of the epithelial cell polarity and allows for the generation of apical-out 3D organoids. Second, we describe how to generate 2-dimensional (2D) monolayers from single cell suspensions derived from intestinal organoids, comprised of&#160;mature and differentiated cell types. These techniques provide novel tools to study apical-specific interactions of the epithelium with external cues in vitro and promote the use of organoids as a platform to facilitate precision medicine.

Here we present two protocols that allow the modeling of intestinal apical-specific interactions. Organoid-derived intestinal monolayers and Air-Liquid Interface (ALI) cultures facilitate the generation of well-differentiated epithelia accessible from both luminal and basolateral sides, whereas polarity-inverted intestinal organoids expose their apical side and are amenable to high throughput assays.

The lining of the gut epithelium is made up of a simple layer of specialized epithelial cells that expose their apical side to the lumen and respond to ...

Rat Intestinal Crypts Isolation and Intestinal Organoids Generation

Generation and Manipulation of Rat Intestinal Organoids

When using organoids to assess physiology and cell fate decisions, it is important to use a model that closely recapitulates in vivo contexts. Accordingly, patient-derived organoids are used for disease modeling, drug discovery, and personalized treatment screening. Mouse intestinal organoids are commonly utilized to understand aspects of both intestinal function/physiology and stem cell dynamics/fate decisions. However, in many disease contexts, rats are often preferred over mice as a model due to their greater physiological similarity to humans in terms of disease pathophysiology. The rat model has been limited by a lack of genetic tools available in vivo, and rat intestinal organoids have proven fragile and difficult to culture long-term. Here, we build upon previously published protocols to robustly generate rat intestinal organoids from the duodenum and jejunum. We provide an overview of several downstream applications utilizing rat intestinal organoids, including functional swelling assays, whole mount staining, the generation of 2D enteroid monolayers, and lentiviral transduction. The rat organoid model provides a practical solution to the need of the field for an in vitro model which retains physiological relevance to humans, can be quickly genetically manipulated, and is easily obtained without the barriers involved in procuring human intestinal organoids.

Author Spotlight: Generation and Manipulation of Rat Intestinal Organoids  

Here, we present a protocol to generate rat intestinal organoids and use them in several downstream applications. Rats are often a preferred preclinical model, and the robust intestinal organoid system fills the need for an in vitro system to accompany in vivo studies.

When using organoids to assess physiology and cell fate decisions, it is important to use a model that closely recapitulates in vivo contexts. ...

Generation of Apical-Out Intestinal Organoids and Assessment of Organoid Proliferation Rate Using EdU-Labeling

Here, we describe the generation of floating cultures of apical-out intestinal organoids from hydrogel-embedded intestinal organoid cultures. Concurrently, floating basal-out organoid cultures are established for direct comparison between apical-out and basal-out organoids. Apical-out and basal-out organoids are subsequently subjected to the thymidine analog 5-ethynyl-2&#39;-deoxyuridine (EdU), which is integrated into the newly synthesized DNA during the S-phase of cell division. This incorporation into DNA can be&#160;visualized&#160;in morphologically intact organoids using laser scanning confocal microscopy. Cells labeled with Hoechst33342 and EdU are then quantified in a semiautomatic manner using image analysis software. Calculation of the percentage of EdU-positive cells of the total number of cells allows for the analysis of cell proliferation in three-dimensional (3D) organoids. Despite being used here for the analysis of proliferation in intestinal organoids, the protocol is applicable to the analysis of nucleus-specific stainings of various sorts in other organoids or two-dimensional cell cultures as well.

Here, we describe a protocol for generating apical-out intestinal organoids from standard matrix-embedded organoid cultures. It also outlines the subsequent incorporation of EdU into actively proliferating cells and the semiautomatic quantification of EdU-positive cells.

Generation of hiPSC-Derived Intestinal Organoids for Developmental and Disease Modelling Applications

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