Thank you to our many collaborators and mentors, including Noah Shroyer, Michael Helmrath, James Wells, and Faranak Fattahi who have allowed us to visit their laboratories and have helped us to refine our protocol over the years. We would also like to thank Chris Mayhew and Amy Pitstick of the Pluripotent Stem Cell Facility and the Center for Stem Cell &#38; Organoid Medicine (CuSTOM) at the Cincinnati Children&#39;s Hospital Medical Center for providing our lab with HIO training, guidance, and advice.&#160;This research was funded by the Texas Medical Center Digestive Diseases Center Pilot/Feasibility grant award (funded in part by NIH/NIDDK P30DK056338) (Speer), the NIDDK (NIH 1K08DK131326-01A1) (Speer), the Men of Distinction award (Speer), and the American Neurogastroenterology and Motility Society (ANMS) Transition Award (Speer).

Human embryonic stem cell (hESC) line H9 was sourced from WiCell (Madison, WI) and all experiments involving hESCs were approved by the UTHealth Houston Stem Cell Research Oversight (SCRO) Committee (protocol #SCRO-23-01). For this protocol, all references to coated plates or wells refer to those prepared with hESC-qualified 3D basement membrane matrix.
1. Cell culture preparation
NOTE: Our lab uses H9 hESCs, but multiple hPSC lines have been used successfully by other labs to generate HIOs and ENCCs, including H9 hESCs9,10,12, H1 hESCs9, hESC line UCSF412, and multiple hiPSC lines such as WTC1112, WTC11 AAVS1-CAG-GCaMP6f9, WTC109,10, WTC10 PHOX2B het (+/Y14X)9, and WTC10 PHOX2B null (Y14X/Y14X)9.
<ol>
	<li>Prepare a 6-well plate by coating each desired well with a 3D basement membrane matrix diluted in an appropriate cell culture medium (dilution factor and instructions for preparation are found in the material data sheet). Coat each desired well with 1 mL per well and incubate at 37 &#176;C for at least 1 h or overnight to set prior to plating cells. 
	NOTE: This protocol may be completed with cells from just one well or scaled up as needed. We recommend coating at least three wells to start to facilitate additional passaging and maintenance of undifferentiated hPSCs. 
	It is important to keep the basement membrane matrix cold while working as most matrices begin to polymerize at room temperature. Coated wells are usable for up to 2-3 weeks when stored at 37 &#176;C. Store with adequate diluent to cover the entire well to prevent the coating from drying out.</li>
	<li>Thaw hPSCs (ideally a low passage number) by briefly swirling in a warm water bath. Under a laminar flow hood, resuspend one vial of cells in 2 mL of stem cell media (Table 1). Aspirate the diluent from one previously coated well in a 6-well dish immediately before plating cells then transfer the entire 2 mL of resuspended cells to the coated well. For maintenance of undifferentiated hPSCs (referred to as maintenance plates), incubate at 37 &#176;C, 5% CO2, and change the media every other day with 2 mL of stem cell media per well.</li>
	<li>Passage the cells when they reach 70-80% confluency. Aspirate the media from the side of the well without disturbing the cells. Add 1 mL of room temperature stem cell dissociation reagent to the well and aspirate it within 1 min, leaving a thin film of liquid over the cells. Incubate the plate for 5 min at 37 &#176;C. 
	NOTE: Waiting until cells are past 80% confluency to passage may cause problems with premature differentiation, which will reduce yield on subsequent steps.</li>
	<li>Add 1 mL per well of warm stem cell medium and tap the side of the plate to dislodge colonies. Gently pipette media using a P1000 micropipette with a wide-mouth pipette tip (see the Table of Materials) to break up the colonies while minimizing cell damage. 
	NOTE: When triturating the cell suspension, it is recommended to break up large clumps of cells but allow smaller clusters to remain intact. It is not recommended to reach a single-cell suspension but rather to aim for fewer, smaller colonies spread out evenly throughout the well.</li>
	<li>Aspirate the diluent from a previously coated well in a 6-well dish and add 2 mL of warm stem cell media. Transfer the desired volume of the cell suspension into this well to maintain hPSC colonies, changing the media every other day with 2 mL of stem cell media per well. 
	NOTE: Our lab typically transfers volumes between 50-150 &#181;L to enable passaging of hPSC maintenance plates once per week. This volume may vary with the specific hPSC line and passage number and thus may require adjustment. Use the remaining cells to initiate ENCC generation (Section 2) or HIO generation (Section 3). Newly thawed hPSCs should be passaged at least twice prior to advancing to either ENCC generation or HIO generation.</li>
</ol>
2. ENCC generation
NOTE: Our method for ENCC generation closely follows that described by the Fattahi lab and used by Schlieve et al.10. We utilize a slightly modified version of the protocol option B through the sections &#34;Enteric Neural Crest (ENC) Induction (Days 0-12)&#34; and &#34;ENCC Spheroid Formation (Day 12-15)&#34; in Barber et al.10,12.
<ol>
	<li>Aspirate the diluent from a previously coated well in a new 6-well dish and add 2 mL of warm stem cell media. Transfer 850 &#181;L of hPSC cell suspension from step 1.5 to the same well (typically a 5:6 passaging ratio).</li>
	<li>Maintain cells at 37 &#176;C, 5% CO2. Change the media every other day with 2 mL of stem cell media per well until confluency reaches 60-80%. Then, the plate is ready to begin ENC induction.</li>
	<li>ENC induction
	<ol>
		<li>Day 0. Aspirate media from the side of the well and add 2 mL of warm Cocktail A media (Table 1). Return to the incubator for 48 h.</li>
		<li>Day 2. Aspirate Cocktail A media from the side of the well and replace with 2 mL of warm Cocktail B media (Table 1). Return to the incubator for 48 h.</li>
		<li>Day 4. Aspirate Cocktail B media from the side of the well and replace with 2 mL of fresh, warm Cocktail B media. Return to the incubator for 48 h.</li>
		<li>Days 6-12. Aspirate Cocktail B media from the side of the well and replace with 2 mL of warm Cocktail C media (Table 1). Return to the incubator and replace media with 2 mL of fresh, warm Cocktail C every 48 h until the completion of ENC induction on Day 12. 
		NOTE: At this stage, ENC lineages may be confirmed by gene expression analysis as described by Barber et al.12.</li>
	</ol>
	</li>
	<li>ENCC spheroid formation
	<ol>
		<li>Day 13. Aspirate Cocktail C media from the side of the well. Add 1 mL of a warm enzymatic cell detachment reagent and incubate at 37 &#176;C for 30 min. Tap the side of the plate to dislodge cells and transfer the cell suspension to a 15 mL conical tube using a wide-mouth pipette tip. Wash the well with 1 mL of Neural Crest Cell (NCC) media (Table 1) and add the washings to the 15 mL conical tube.</li>
		<li>Centrifuge the cell suspension in the 15 mL conical tube at 300 &#215; g for 1 min. Aspirate the supernatant. Gently resuspend the pellet in 2 mL of warm NCC media.</li>
		<li>Add the resuspended cells to a new, uncoated well of an ultra-low-binding plate and incubate at 37 &#176;C, 5% CO2 for 48 h.</li>
		<li>Days 15-21. Monitor ENCC spheroid growth and replace media with 2 mL of fresh, warm NCC media every 48 h. 
		NOTE: Media changes should be performed with care to avoid damaging or aspirating the ENCC spheroids. This is best accomplished by swirling the plate in a circular motion to focus the spheroids in the middle of the well, then aspirating the media from the edge of the well. 
		ENCC spheroid formation is typically complete by day 15 though it is acceptable to culture them up to day 21 to accommodate ideal timing of coculture with mid-hindgut spheroids.</li>
	</ol>
	</li>
</ol>
3. HIO generation (mid-hindgut spheroid phase)
NOTE: Our method for HIO generation is fundamentally the same as the original protocol described in detail by McCracken et al. and others from the Spence and Wells labs6,7. The process occurs in two phases: Definitive endoderm (DE) induction and Mid-hindgut spheroid formation.
<ol>
	<li>Preparation for HIO Generation
	<ol>
		<li>Prepare a 24-well plate by coating each desired well with a 3D basement membrane matrix diluted in an appropriate cell culture medium (dilution factor and instructions for preparation are found in the material data sheet). Coat each desired well with 500 &#181;L per well and incubate at 37 &#176;C for at least 1 h or overnight to set prior to plating cells. 
		NOTE: It is important to keep the basement membrane matrix cold while working as most matrices begin to polymerize at room temperature. Coated wells are usable for up to 2-3 weeks when stored at 37 &#176;C. Store with enough diluent to cover the entire well to prevent the coating from drying out. 
		&#8203;Plan to coat 6 wells of a 24-well plate for each 1 well from the 6-well hPSC maintenance plates described above. This ratio may be scaled up as desired.</li>
		<li>Aspirate the diluent from all previously coated wells in a 24-well dish. Add sufficient warm stem cell media to the hPSC cell suspension from step 1.5 and distribute 500 &#181;L into each well (e.g., if 850 &#181;L of hPSC cell suspension is left after passaging the hPSC maintenance plate, then for 6 wells of a 24-well plate, mix 2,150 &#181;L of warm stem cell media with 850 &#181;L of the hPSC cell suspension from step 1.5, and 500 &#181;L distributed in each of 6 wells). 
		NOTE: As we typically passage hPSC maintenance plates with volumes of 50-150 &#181;L, we usually have volumes of 850-950 &#181;L available for this step. Thus, the volume of hPSCs per well in the 24-well plate approximates 150 &#181;L/well. 
		&#8203;Once the newly prepared 24-well plate has been placed in the incubator, gently agitate the plate back and forth (north to south and east to west) to distribute the cells as evenly as possible across the bottom of the well.</li>
		<li>Maintain cells at 37 &#176;C, 5% CO2. Change the media every other day with 500 &#181;L of stem cell media per well until confluency reaches 50-70%. Then, the plate is ready to begin DE induction. 
		NOTE: It is critical that cells do not reach &#62;70% confluency prior to DE induction, as higher confluency adversely affects endoderm density and hindgut morphogenesis in subsequent stages. Ideally, cells reach approximately 50-70% confluency after 24-48 h at the specified passage ratios.</li>
	</ol>
	</li>
	<li>DE induction
	<ol>
		<li>Day 1. Once hPSCs have reached 50-70% confluency, aspirate the stem cell media from each well completely and replace with 500 &#181;L of warm Day 1 DE media (Table 1) per well. Incubate at 37 &#176;C, 5% CO2 for 24 h.</li>
		<li>Day 2. Aspirate the Day 1 DE and replace with 500 &#181;L of warm Day 2 DE (Table 1) media per well. Incubate at 37 &#176;C, 5% CO2 for 24 h.</li>
		<li>Day 3. Aspirate the Day 2 DE and replace with 500 &#181;L of warm Day 3 DE (Table 1) media per well. Incubate at 37 &#176;C, 5% CO2 for 24 h.</li>
	</ol>
	</li>
	<li>Mid-hindgut spheroid formation
	<ol>
		<li>Days 4-8. Aspirate the media from each well completely and replace with 500 &#181;L of warm Mid-hindgut media (MHGM, Table 1) per well. Incubate at 37 &#176;C, 5% CO2. Repeat this step daily until Day 8, at which time mid-hindgut spheroids are ready for collection and coculture (Section 4).</li>
	</ol>
	</li>
</ol>
4. Co-culture of mid-hindgut spheroids with ENCCs
NOTE: A graphic summary of this section is provided in Figure 1.
<ol>
	<li>Preparation of ENCC suspension
	<ol>
		<li>Collect Day 15-21 ENCC spheroids from step 2.4.4. by carefully removing the media from the well, avoiding aspirating the spheroids using the swirl method described in the note for that step.</li>
		<li>Add 1 mL of a warm enzymatic cell detachment reagent to the well and incubate at 37 &#176;C for 30 min.</li>
		<li>Tap the side of the plate to continue dissociating spheroids and triturate with a P1000 micropipette with a wide-mouth tip by pipetting gently up and down to break up clumps of cells until the mixture is homogenous.</li>
		<li>Transfer the ENCC suspension to a 15 mL conical tube. Wash the well with 1 mL of warm NCC media (see Table 1) to collect any remaining cells and add this to the 15 mL conical tube.</li>
		<li>Centrifuge the cell suspension at 300 &#215; g for 1 min. Aspirate the supernatant with a sterile pipette tip.</li>
		<li>Using a wide-mouth pipette tip, resuspend the cell pellet in 1 mL of warm NCC media. Calculate the concentration of the ENCC suspension using a hemacytometer or cell counter.</li>
	</ol>
	</li>
	<li>Combining ENCC suspension with mid-hindgut spheroids
	<ol>
		<li>Collect the mid-hindgut spheroids from all wells of an HIO plate using a wide-mouth pipette tip and transfer to a 15 mL conical tube.</li>
		<li>Determine the number of coculture wells that will be used within a 24-well plate. Aim to plate 10-30 mid-hindgut spheroids and 50,000-100,000 ENCCs per well for coculture. 
		NOTE: In practice, there is often a one-to-one relationship between the number of wells with healthy spheroids and the final number wells that will be needed for coculture. A hemocytometer can also be utilized to calculate the concentration of mid-hindgut spheroids collected.</li>
		<li>Add the calculated volume of ENCC suspension to deliver 50,000-100,000 ENCCs per planned coculture well to the conical tube with the mid-hindgut spheroids. Gently triturate with a wide-mouth pipette tip to mix the mid-hindgut spheroids and ENCCs uniformly.</li>
		<li>Centrifuge at 300 &#215; g for 1 min and carefully aspirate the supernatant with a sterile pipette tip. Using a wide-mouth pipette tip, resuspend the cell pellet in a volume of cold, phenol-free (clear), growth factor-reduced basement membrane matrix, equal to 30 &#181;L per final coculture well. 
		NOTE: Avoid forming bubbles in the clear basement&#160;membrane matrix during resuspension, as these are challenging to remove, will impede proper imbibition of the media by the HIO+ENCC cocultures, and will reduce visualization of the HIO+ENCC cocultures as they grow.</li>
		<li>To the center of each dry well, pipette a 30 &#181;L droplet of resuspended mid-hindgut spheroids + ENCCs in a clear basement membrane matrix without any surrounding media. Once all droplets are plated in the wells, cover and invert the plate. Incubate inverted at 37 &#176;C, 5% CO2 for 30 min. 
		NOTE: This step allows time for the mid-hindgut spheroids and ENCCs to fall away from the bottom of the plate and become suspended during polymerization of the clear 3D basement&#160;membrane matrix.</li>
	</ol>
	</li>
	<li>In Vitro HIO+ENS coculture
	<ol>
		<li>Day 0: Once the clear basement membrane matrix has polymerized, cover each droplet in 500 &#181;L of warm Day 0-3 HIO media (see Table 1).</li>
		<li>Day 3: Carefully aspirate the Day 0-3 HIO media from the side of the well, without disturbing the clear basement membrane matrix droplet. Replace the media with 500 &#181;L of warm Day 3-14 HIO media (see Table 1). Return the plate to the incubator. At this stage, change the media every 3-4 days.</li>
		<li>Days 7-14: Continue replacing Day 3-14 HIO media as needed every 3-4 days and monitor the growth.</li>
	</ol>
	</li>
	<li>Splitting HIOs
	<ol>
		<li>Day 14: Examine each well carefully under a microscope and assess how many developing HIO + ENS are in each clear 3D basement membrane matrix droplet.</li>
		<li>Prepare 2 mL microcentrifuge tubes with 30 &#181;L of clear basement membrane matrix and keep these on ice.</li>
		<li>Under a laminar flow hood, use fine forceps to carefully remove the clear 3D basement membrane matrix droplet with the suspended HIO+ENS. Gently tease apart individual HIO+ENS. 
		NOTE: Surgical loupes or a dissecting microscope can be very helpful at this stage but are not strictly necessary as HIOs are often grossly visible to the naked eye.</li>
		<li>Place each isolated HIO + ENS into one of the prepared microcentrifuge tubes containing 30 &#181;L of clear basement membrane matrix using either forceps or a wide-mouth pipette tip. 
		NOTE: Old clear 3D basement membrane matrix does not need to be liquified or dissolved at this stage. Avoid disrupting the HIOs if possible, though if this occurs it may still grow through later stages.</li>
		<li>Repeat step 4.2.5 for the split and replated HIO+ENS. Once the clear 3D basement membrane droplets are polymerized, add 500 &#181;L of warm, fresh Day 3-14 HIO&#160;media to each well and incubate overnight.</li>
	</ol>
	</li>
	<li>HIO+ENS coculture continued
	<ol>
		<li>Day 15: Carefully aspirate the Day 3-14 HIO media from the side of the well. Replace the media with 500 &#181;L warm Day 15-28 HIO&#160;media (see Table 1).</li>
		<li>Day 15-40: Continue replacing Day 15-28 HIO media as needed every 3-4 days and monitoring HIO + ENCC growth.</li>
	</ol>
	</li>
</ol>

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The authors do not have any conflicts of interest to disclose.

HIOs have been used as a model system for human intestinal development since the early 2010&#39;s and have become increasingly more complex since then. It is now possible to provide these constructs with an ENS, allowing for new opportunities in the study of normal development that may be applied to understanding and better treating a number of clinical gastrointestinal entities.
In vivo transplantation 
ENCCs integrate into the mid-hindgut spheroids and form an ENS during the in vitro coculture phase. However, several groups, including ours, elect to transplant the HIO + ENS into a living host after day 28 to facilitate additional growth and maturation supported by host vasculature. We typically transplant HIO + ENS in vivo between days 28 and 40 of in vitro growth. However, there are other labs that have grown HIOs longer than 40 days in vitro. As the transcriptome in those longer in vitro cultures remained fetal-like without further maturation13, coculturing in vitro beyond 40 days affords no further advantage. Methods and recommendations for optimizing transplantation procedures have been reported by multiple groups, including ours 9,10,14,15,16,17,18,19,20. We currently favor transplantation beneath the kidney capsule as first reported by Watson et al. in 2014 and described in detail by Mahe et al. in 2017 for HIO+ENS as this has demonstrated the most robust tHIO engraftment rates in our hands16,19. We have harvested transplanted HIO + ENS as early as 4 weeks and as late as 20 weeks for analysis depending on the application.
Critical steps 
Newly thawed hPSCs should be cultured for at least 2 weeks prior to initiation of ENCC generation or HIO differentiation. At each step of the protocol, it is imperative to ensure the cell confluence is within the recommended range. A common bottleneck is the mid-hindgut spheroid generation stage, which in our hands, seems to be very sensitive to fluctuations in confluency of the DE, temperature, CO2, and other extrinsic factors.
Once mid-hindgut spheroids are formed, we recommend splitting HIOs to reduce competition, improve nutrient delivery, and thereby maximize growth potential of each HIO. This is especially beneficial in crowded wells with three or more HIOs. We typically pick the best HIO + ENS with good growth (Figure 4) to isolate and reduce competition but save as many as possible. We have found that 14 days is the best time point to split HIOs since they are grossly visible and have had adequate time to allow ENCC integration into the mesenchyme.
Modifications and troubleshooting 
Each plate should be monitored daily to ensure progress through the expected phases of differentiation and morphology as described above. If at any stage, the cells are not behaving as expected, there are some common pitfalls that could be contributing. First, ensure that the cell confluence is at recommended levels at the initiation of each step. Colonies that are too dense are prone to premature differentiation, morphologic abnormalities, and cell death. Colonies that are too sparse can also demonstrate morphologic differences but may result in poor yields and wasted reagents. Second, many of the growth factors, active ingredients, and reagents used at various steps of this protocol have limited shelf lives. Most should be aliquoted immediately upon receipt and kept at -80 &#176;C for long-term storage. Aliquots may only be stable at 4 &#176;C for up to 2 weeks. Ensure all reagents are stored according to manufacturer guidelines and keep records of all lot numbers while reagents are in use. During troubleshooting, it may be necessary to run duplicate experiments with a new reagent and an older aliquot that was successful recently or to replace a reagent. Finally, best practice cell culture techniques should be applied as many steps in this protocol are highly influenced by extrinsic factors. Regular monitoring of incubator CO2 levels, ensuring strict measures to prevent contamination, regular testing for mycoplasma, and minimizing cell stress from over-frequent media changes or prolonged periods outside the incubators should be prioritized at all times.
Limitations 
One limitation of the protocol described here is that it has only been reproduced using H9 hESCs in our hands. While other groups have utilized other hPSC lines in similar applications, as noted above, we cannot specifically comment on their use for this protocol nor whether one line or another would be superior at certain stages. This protocol also depends on commercially available stem cell media and other products, the exact formulation of which is proprietary and subject to change, which may affect results.
Significance and future work 
In this article, we have presented the method by which our group innervates HIOs, building on the work of several other labs as reviewed and credited above. The aim is to maximize the strengths of each previous method. Our observations have led us to favor a period of early coculture of gut spheroids with ENCCs to expose these tissues to each other and promote signaling crosstalk that likely mimics normal development and promotes more mature cell and HIO morphology. Using this method, we have had consistent success with recent tHIO engraftment rates of &#62;85% for the kidney capsule (N = 12/14 for tHIOs (86%), N = 15/16 for tHIOs + ENS (94%), N = 27/30 overall for tHIOs &#177; ENS (90%)), and ENS integration rates of 100% for tHIOs engrafted (N = 15/15 tHIOs + ENS). While our ENS integration rates are successful, we have discovered that there is variability in the development and maturation of the ENS present within tHIOs.
Our future studies are focused on reducing variability and improving ENS development such that spatial organization and cellular maturity are more reproducible. Future studies will also continue this iterative process and focus on new ways to optimize these methods to better recapitulate normal intestinal development and eventually allow for the recreation of morphologically and functionally mature intestinal tissue as a therapy for human patients with intestinal loss or dysfunction.

The human small intestine is a complex, multilayered organ that carries out numerous essential functions such as digestion, nutrient absorption, fluid regulation, immune barrier function, and motility. Numerous clinical diseases, such as short bowel syndrome, enteropathies, or motility disorders, are characterized by critical reduction of intestinal mass or disruptions of normal physiology leading to significant morbidity and mortality1,2,3,4. Current treatment options often include surgery to remove the dysfunctional intestine at the cost of decreased intestinal length and, thereby, the functional capacity of the remaining bowel5. There is a need for regenerative therapies that are additive in nature, increasing functional gut capacity and restoring intestinal function in ways current therapeutic paradigms are unable to achieve.
Bioengineered small intestine is a promising solution to these challenges. Human intestinal organoids (HIOs) derived from human pluripotent stem cells (hPSCs) are one starting material for the bioengineered small intestine. In 2011, Spence et al. first reported the successful creation of modern HIOs from hPSC lines, including both H1 and H9 human embryonic stem cell (hESC) and multiple human induced pluripotent stem cell (hiPSC) lines6,7. Their protocol included a carefully timed series of incubations with specific growth factors to mimic human fetal intestinal development. Activin A, a TGF&#946; signaling molecule, drives hPSC differentiation towards endodermal fate followed by directed posterior patterning with Wnt/FGF. Under these conditions, hPSCs self-organize to form gut spheroids containing polarized epithelium and mesenchyme. 3D culture within a basement membrane matrix allows for additional development resulting in organoids with an internal lumen, villus-like involutions of epithelium, and self-regenerating progenitor cell niches within crypt-like structures.
While HIOs generated using this original 2011 protocol structurally resemble native intestine, they lack the capacity to generate the neurons or glia of the enteric nervous system (ENS). In 2017, two major papers described similar but distinct methods of innervating HIOs. ENS progenitors (enteric neural crest cells, ENCCs) can be derived from hPSCs. In culture, ENCCs form 3D neurospheres, which can differentiate into enteric neurons and glia under appropriate conditions. Workman and Mahe et al. modified an existing protocol to derive ENCCs from hPSCs and treated them with FGF-enriched media followed by retinoic acid for 2 days for posteriorization and promotion of vagal fate8,9. Day 6 neurospheres were incubated for another 4 days without RA, and then migrated cells were collected after enzymatic detachment. Approximately 20,000-50,000 ENCCs were aggregated with early mid-hindgut spheroids and these ENCC-seeded gut spheroids were grown in vitro in a clear 3D basement membrane matrix for 28 days until in vivo engraftment into the kidney capsule of immunodeficient mice for 6-10 weeks. The resulting HIO + ENS demonstrated significant maturation of epithelial and mesenchymal components as well as neuroglial structures similar to ganglia, albeit with lower cell body density than native intestine, absence of certain clinically relevant neuronal subtypes (i.e., CHAT-positive neurons in HIO + ENS after transplantation), and overall fetal-like characteristics.
The same year, Schlieve et al. published an alternative method involving a mixture of 40-60 intact, day 15 ENCC neurospheres with more mature HIOs at the time of in vivo transplantation into the omentum of immunodeficient mice for 3 months without prior in vitro co-culture10. Their constructs, called ENCC-HIO-TESI (tissue-engineered small intestine), appeared to contain a more mature ENS phenotype than that of Workman and Mahe&#39;s HIO + ENS with greater neuronal subtype diversity and neuroepithelial synaptic connections not seen in HIO + ENS. Importantly, the ENCCs used in Schlieve&#39;s experiments were derived via a different method, as described previously by Fattahi et al.11. Briefly, hPSCs (both hESCs and hiPSCs) underwent neural crest induction in FGF2-enriched media. These cells were also treated with RA to establish an enteric vagal fate, but for 5 days (days 6-11), an increase from Workman and Mahe&#39;s 2 days (day 4-5). In 2019, Barber et al. published a revised version of the Fattahi protocol including a more thorough description of culture conditions and a transition to defined basal media to reduce inconsistency and reflect changing cell culture preferences at the time12.
Our method for innervating HIOs, described in detail below, incorporates elements of both the Workman/Mahe and Schlieve protocols. While the ENS in Schlieve&#39;s ENCC-HIO-TESI appeared more mature with greater neuronal diversity and neuroglial integration, both methods successfully integrated a functional ENS into the HIOs with demonstrated changes in gastrointestinal transcriptional expression. Workman and Mahe&#39;s HIO + ENS induced increased expression of several genes related to intestinal stem cells and epithelial cell development, which were not altered in ENCC-HIO-TESI. One possible explanation for these distinctions is the different time points when ENCCs and HIOs were combined between the protocols. Our lab has observed that early coculture results in the increased expression of multiple genes involved in epithelial and mesenchymal differentiation, and the timing of coculture influences epithelial cell diversity (unpublished data). It is possible that earlier exposure of ENS precursors to developing HIOs and vice versa provides time for yet undefined signaling crosstalk that promotes epithelial diversity and other early developmental processes.

<table><tbody><tr><td>100x Non-Essential Amino Acids Solution (NEAA)</td><td>ThermoFischer Scientific</td><td>11140050</td><td></td></tr><tr><td>15 mL conical tubes</td><td>Thermo Scientific</td><td>12565269</td><td></td></tr><tr><td>Accutase</td><td>STEMCELL Technologies</td><td>07920</td><td>&quot;enzymatic cell detachment reagent&quot;</td></tr><tr><td>B-27 Supplement (50x), minus vitamin A</td><td>ThermoFischer Scientific</td><td>12587010</td><td>For ENCC</td></tr><tr><td>B-27 Supplement (50x), serum free</td><td>Gibco</td><td>17504044</td><td>For HIO</td></tr><tr><td>Bright-Line Hemacytometer</td><td>Hausser Scientific</td><td>3120</td><td></td></tr><tr><td>Corning Costar Ultra-Low Attachment Microplates</td><td>Fisher Scientific</td><td>07-200-601</td><td></td></tr><tr><td>Corning Flat-Bottom Plate 24-well, TC treated</td><td>VWR</td><td>29442-044</td><td></td></tr><tr><td>Corning Flat-Bottom Plate 3516 6 well</td><td>VWR</td><td>29442-042</td><td></td></tr><tr><td>Essential 6 Media</td><td>Thermo Fischer</td><td>A1516401</td><td></td></tr><tr><td>Essential 8 Media</td><td>Thermo Fischer</td><td>A2858501</td><td>Alternative stem cell media, used for ENCC plates.&amp;nbsp;</td></tr><tr><td>Fine-tip forceps</td><td>Dumont</td><td>11223-20</td><td></td></tr><tr><td>Forma Steri-Cycle i160</td><td>Thermo Scientific</td><td>50145522</td><td></td></tr><tr><td>Gibco Advanced DMEM/F12</td><td>ThermoFischer Scientific</td><td>12634-010</td><td></td></tr><tr><td>Gibco HEPES 1 M</td><td>ThermoFischer Scientific</td><td>15630-080</td><td></td></tr><tr><td>Gibco Neurobasal Medium</td><td>ThermoFischer Scientific</td><td>21103-049</td><td></td></tr><tr><td>Glutagro, 200 mM, 100x</td><td>Corning</td><td>25-015-CI</td><td></td></tr><tr><td>Glutamax</td><td>ThermoFischer Scientific</td><td>35050061</td><td></td></tr><tr><td>H9 human ESC</td><td>Wicell International Stem Cell Bank</td><td>N/A</td><td></td></tr><tr><td>HyCloneTM FBS Defined</td><td>VWR</td><td>16777-002</td><td></td></tr><tr><td>LabGard Biological Safety Cabinet</td><td>Nuaire</td><td>Nu-430-400</td><td></td></tr><tr><td>Matrigel GFR Basement Membrane Matrix, Phenol Red-Free, LDEV-Free</td><td>Corning</td><td>356231</td><td>&quot;Clear 3D basement membrane matrix&quot;</td></tr><tr><td>Matrigel hESC-Qualified Matrix, LDEV-Free</td><td>Corning</td><td>354277</td><td>&quot;3D basement membrane matrix&quot;</td></tr><tr><td>Micropipettes</td><td>Eppendorf (100-1000, 20-200, 10-100, 2-20, 0.5-10, 0.1-2.5 uL)</td><td>2231300008</td><td></td></tr><tr><td>mTeSR 1</td><td>STEMCELL Technologies</td><td>85850</td><td>&quot;stem cell media&quot;&lt;br/&gt; Catalog number includes the 5x supplement, to be added in bulk in advance.&amp;nbsp;</td></tr><tr><td>N-2 Supplement (100x)</td><td>Gibco</td><td>17502048</td><td>For HIO</td></tr><tr><td>N-2 Supplement, CTS (Cell Therapy Systems)</td><td>Thermo Fisher</td><td>A1370701</td><td>For ENCC. Slightly different formulation.</td></tr><tr><td>Nikon DS-Fi2 TS-100 microscope</td><td>Nikon</td><td>TS100</td><td></td></tr><tr><td>Noggin-conditioned media</td><td>Texas Medical Center Digestive Disease Center GEMS Core, Enteroid/Organoid Sub-core</td><td>N/A</td><td></td></tr><tr><td>Penicillin-Streptomycin (10,000 U/mL)</td><td>ThermoFischer Scientific</td><td>15140-122</td><td></td></tr><tr><td>Recombinant Human Activin A</td><td>Cell Guidance Systems</td><td>GFH6-100</td><td></td></tr><tr><td>Recombinant Human BMP-4</td><td>Fisher Scientific</td><td>314BP010</td><td></td></tr><tr><td>Recombinant Human EGF Protein, CF</td><td>ThermoFisher Scientific</td><td>236-EG-200</td><td></td></tr><tr><td>Recombinant Human FGF basic/FGF2 (146 aa) Protein</td><td>ThermoFischer Scientific</td><td>233-FB-010</td><td></td></tr><tr><td>Recombinant Human FGF-4</td><td>Peprotech</td><td>100-31</td><td></td></tr><tr><td>ReLeSR</td><td>STEMCELL Technologies</td><td>5872</td><td>&quot;Stem cell dissociation reagent&quot;</td></tr><tr><td>Retinoic acid</td><td>SIGMA</td><td>R2625-50MG</td><td></td></tr><tr><td>Rnase-free Microfuge tubes, 2 mL</td><td>Thermo Scientific</td><td>AM12425</td><td></td></tr><tr><td>RPMI 1640 Medium</td><td>ThermoFischer Scientific</td><td>11875093</td><td></td></tr><tr><td>R-Spondin conditioned media</td><td>Texas Medical Center Digestive Disease Center GEMS Core, Enteroid/Organoid Sub-core</td><td>N/A</td><td></td></tr><tr><td>SB 431542, Tocris Bioscience</td><td>Fisher Scientific</td><td>16-141-0</td><td></td></tr><tr><td>Sorvall ST 16R Centrifuge</td><td>Thermo Scientific</td><td></td><td></td></tr><tr><td>Standard Wide Orifice Pipettor Tips</td><td>VWR</td><td>89049-166</td><td></td></tr><tr><td>Stemolecule Chir99021 in Solution</td><td>Stemgent</td><td>04-0004-02</td><td></td></tr><tr><td>Sterile filter pipette tips</td><td>VWR (1000uL, 200uL, 10uL)</td><td>76322-154, 76322-150, 89174-520</td><td></td></tr><tr><td>Vitronectin XF</td><td>Stem Cell Technologies</td><td>7180</td><td>Alternative 3D basement membrane matrix, used for ENCC plates.&amp;nbsp;</td></tr></tbody></table>

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.

The ENS regulates the essential functions of the mature small intestine, including peristalsis, nutrient absorption, fluid transport, and epithelial barrier maintenance. Thus, the goal of innervating HIOs is to provide these constructs with the elements needed to develop more mature, higher-level functionality. To this end, our lab specifically studies the development of the ENS within HIOs as well as the functional outcomes at different stages.
It is important to monitor cell growth, differentiation, and morphogenesis over time to ensure that each step is progressing as expected before proceeding to the next stage. ENCC differentiation and spheroid formation occur over a period of up to 3 weeks, with representative brightfield micrographs of ENCCs after differentiation and at different stages of ENCC spheroid formation shown in Figure 2. ENCCs should start to form visible aggregates within the cell monolayer by day 12 followed by spontaneous spheroid formation after replating on the ultra-low-attachment plates. ENCC spheroids tend to grow rapidly at this stage and are easily visible to the naked eye.
During the HIO generation steps. it is normal to observe some cell death after Day 1 of DE induction. Proliferation should resume on Days 2 and 3, and ultimately by the end of Day 3 confluence should approach 100%. Cells should appear large and flat with clear borders and fairly homogeneous appearance at the end of DE induction. As mid-hindgut spheroids begin to form, many will be tethered to the monolayer by a fragile &#34;chain&#34; of cells and some will spontaneously detach and float freely in the media. Representative brightfield micrographs of DE at the end of the induction phase and day 8 mid-hindgut spheroids ready for coculture are shown in Figure 3.
After coculture of ENCCs with mid-hindgut spheroids, resulting HIO + ENS will grow significantly and develop visible mesenchyme characterized by thin, hazy projections that form a halo-like border around internal epithelial involutions and luminal structures. Representative brightfield micrographs demonstrate the growth of HIO + ENS cocultures over time (Figure 4).
We have shown that ENCCs cocultured with early mid-hindgut spheroids by the method described above migrate towards HIOs, integrate within the surrounding mesenchyme, and differentiate to form enteric neurons and glia. Serial fluorescent imaging of HIOs cocultured with ENCC derived from GFP-labeled hESCs showed that ENCCs survive in vitro within the HIO even without the addition of exogenous growth factors to support ENCC growth (such as glial cell-derived neurotrophic factor or GDNF), suggesting that ENCCs were successfully integrated into the HIO and received adequate survival and differentiation signals from the mesenchyme (Figure 5). When cocultured HIO + ENS were transplanted into immunodeficient mice and allowed to grow in vivo for 4-20 weeks (see section In vivo transplantation below), immunohistochemistry confirmed the expression of neuronal markers such as TUJ1 (Figure 6) and HuC/D (Figure 7A) as well as glial markers such as S100&#946; (Figure 7B), confirming that ENCCs can differentiate to form both neurons and glia required for normal function.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/67702/67702fig01.jpg" /> 
Figure 1: Summary of HIO + ENCC coculture method. Directed differentiation of hPSCs generates a population of ENCC, which then self-organize into ENCC spheroids in the presence of FGF2 and other supplements. ENCC spheroids are dissociated to create a single-cell suspension between days 15-21. Separately, hPSCs undergo induction with Activin A to form a monolayer of DE. This is then directed to form mid-hindgut spheroids, which will eventually grow into HIOs once suspended in 3D clear&#160;basement membrane matrix droplets. After 4 days of mid-hindgut spheroid formation (total day 8 from initiation of DE induction), mid-hindgut spheroids are combined with the ENCC single-cell suspension and plated in a droplet of clear basement membrane matrix. HIO + ENCC cocultures are grown in vitro for another 28-40 days before transplanting in vivo into immunosuppressed mice for further development and maturation. Abbreviations: ENCCs = enteric neural crest cells; HIO = human intestinal organoid; hPSCs = human pluripotent stem cells; FGF2 = fibroblast growth factor 2; DE = definitive endoderm. <a href="https://www.jove.com/files/ftp_upload/67702/67702fig01large.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/67702/67702fig02.jpg" /> 
Figure 2: Representative results for ENCC spheroid generation. On day 12, cells have completed the ENCC differentiation stage. They are collected, washed, transferred to NCC media, and replated in ultra-low-attachment plates. ENCCs self-organize into spheroids over the next several days and these are typically ready for coculture by days 15-21. Example brightfield images show cells at the end of ENCC differentiation, early spheroid formation at Day 14 and fully formed ENCC spheroids suitable for coculture at Day 18. Scale bar = 1 mm. Abbreviations: ENCCs = enteric neural crest cells; NCC = Neural crest cell. <a href="https://www.jove.com/files/ftp_upload/67702/67702fig02large.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/67702/67702fig03.jpg" /> 
Figure 3: Representative results for mid-hindgut spheroid generation. hESCs undergo DE induction for 3 days, after which cells form a homogeneous monolayer in the well. After 4 days of incubation with Mid-Hindgut media containing FGF4/Chiron, cells form mid-hindgut spheroids that begin to detach from the monolayer in the well. Scale bar = 1 mm. Abbreviations: hESCs = human embryonic stem cells; DE = definitive endoderm; FGF4 = fibroblast growth factor 4. <a href="https://www.jove.com/files/ftp_upload/67702/67702fig03large.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/67702/67702fig04.jpg" /> 
Figure 4: Progression of HIO + ENS coculture growth over time. Epithelial development is demonstrated by the internal involutions and looping structures labeled with white arrowheads. HIO + ENCC cocultures also develop surrounding mesenchyme, as seen by the hazy, less dense material in the periphery surrounding the epithelium labeled with the black arrowheads. Scale bar = 1 mm. Abbreviations: ENCCs = enteric neural crest cells; HIO = human intestinal organoid. <a href="https://www.jove.com/files/ftp_upload/67702/67702fig04large.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/67702/67702fig05.jpg" /> 
Figure 5: Phase contrast micrographs. (A) HIO + ENCC in vitro. (B) Fluorescence micrograph showing ENCCGFP+ migrating into&#160;HIO + ENCC. (C) Merged images of phase contrast and fluorescence micrographs. Arrows indicate ENCCGFP+ cells. Scale bar = 100 &#181;m. Abbreviations: ENCCs = enteric neural crest cells; HIO = human intestinal organoid. <a href="https://www.jove.com/files/ftp_upload/67702/67702fig05large.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/67702/67702fig06.jpg" /> 
Figure 6: Survival and differentiation of ENCCs into enteric neurons. Pan-neuronal marker neuron-specific beta tubulin III (TUJ1) is seen in 28 days old (B) HIOs + ENCCs but not (A) HIOs without ENCCs in vitro; and in (D) tHIOs + ENCCs after 6.6 weeks in vivo but not (C) tHIOs without ENCCs. E-cadherin (ECAD) marks the intestinal epithelium and TUJ1 marks the enteric neurons. Smooth muscle actin (SMA) marks the smooth muscle of the muscularis mucosae and propria. Scale bar = 100 &#181;m. Abbreviations: ENCCs = enteric neural crest cells; HIO = human intestinal organoid; tHIO = transplanted HIO. <a href="https://www.jove.com/files/ftp_upload/67702/67702fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/67702/67702fig07.jpg" /> 
Figure 7: ENCC differentiation and integration into the tHIO mesenchyme. Formation of (A) neuronal and (B) glial lineages. Neuronal RNA-binding proteins HuC/D (HuC/D) mark enteric neurons. S100 calcium-binding protein &#946; (S100&#946;) marks enteric glia. Scale bars = 100 &#181;m. Abbreviations: ENCCs = enteric neural crest cells; HIO = human intestinal organoid; tHIO = transplanted HIO. <a href="https://www.jove.com/files/ftp_upload/67702/67702fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 1: Composition of media used in this protocol. <a href="https://www.jove.com/files/ftp_upload/67702/Table 1 Media Composition (1).xlsx" target="_blank">Please click here to download this Table.</a>

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.

Innervation of Human Intestinal Organoids

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

innervation-of-human-intestinal-organoids

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

374 Views.  The University of Texas Health Science Center at Houston. 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. 

Video: Innervation of Human Intestinal Organoids

Generation of iPSC-derived Human Brain Organoids to Model Early Neurodevelopmental Disorders

The restricted availability of suitable in vitro models that can reliably represent complex human brain development is a significant bottleneck that limits the translation of basic brain research into clinical application. While induced pluripotent stem cells (iPSCs) have replaced the ethically questionable human embryonic stem cells, iPSC-based neuronal differentiation studies remain descriptive at the cellular level but fail to adequately provide the details that could be derived from a complex, 3D human brain tissue. 
This gap is now filled through the application of iPSC-derived, 3D brain organoids, "Brains in a dish," that model many features of complex human brain development. Here, a method for generating iPSC-derived, 3D brain organoids is described. The organoids can help with modeling autosomal recessive primary microcephaly (MCPH), a rare human neurodevelopmental disorder. A widely accepted explanation for the brain malformation in MCPH is a depletion of the neural stem cell pool during the early stages of human brain development, a developmental defect that is difficult to recreate or prove in vitro. 
To study MCPH, we generated iPSCs from patient-derived fibroblasts carrying a mutation in the centrosomal protein CPAP. By analyzing the ventricular zone of microcephaly 3D brain organoids, we showed the premature differentiation of neural progenitors. These 3D brain organoids are a powerful in vitro system that will be instrumental in modeling congenital brain disorders induced by neurotoxic chemicals, neurotrophic viral infections, or inherited genetic mutations.

Modeling human brain development has been hindered due to the unprecedented complexity of neural epithelial tissue. Here, a method for the robust generation of brain organoids to delineate early events of human brain development and to model microcephaly in vitro is described.

The restricted availability of suitable in vitro models that can reliably represent complex human brain development is a significant bottleneck that ...

Immuno-fluorescent Labeling of Microtubules and Centrosomal Proteins in Ex Vivo Intestinal Tissue and 3D In Vitro Intestinal Organoids

The advent of 3D in vitro organoids that mimic the in vivo tissue architecture and morphogenesis has greatly advanced the ability to study key biological questions in cell and developmental biology. In addition, organoids together with recent technical advances in gene editing and viral gene delivery promises to advance medical research and development of new drugs for treatment of diseases. Organoids grown in vitro in basement matrix provide powerful model systems for studying the behavior and function of various proteins and are well suited for live-imaging of fluorescent-tagged proteins. However, establishing the expression and localization of the endogenous proteins in ex vivo tissue and in in vitro organoids is important to verify the behavior of the tagged proteins. To this end we have developed and modified tissue isolation, fixation, and immuno-labeling protocols for localization of microtubules, centrosomal, and associated proteins in ex vivo intestinal tissue and in in vitro intestinal organoids. The aim was for the fixative to preserve the 3D architecture of the organoids/tissue while also preserving antibody antigenicity and enabling good penetration and clearance of fixative and antibodies. Exposure to cold depolymerizes all but stable microtubules and this was a key factor when modifying the various protocols. We found that increasing the ethylenediaminetetraacetic acid (EDTA) concentration from 3 mM to 30 mM gave efficient detachment of villi and crypts in the small intestine while 3 mM EDTA was sufficient for colonic crypts. The developed formaldehyde/methanol fixation protocol gave very good structural preservation while also preserving antigenicity for effective labeling of microtubules, actin, and the end-binding (EB) proteins. It also worked for the centrosomal protein ninein although the methanol protocol worked more consistently. We further established that fixation and immuno-labeling of microtubules and associated proteins could be achieved with organoids isolated from or remaining within the basement matrix.

Immuno-fluorescent Labeling of Microtubules and Centrosomal Proteins in Ex Vivo Intestinal Tissue and 3D In Vitro Intestinal Organoids

We present protocols for isolation of intestinal 3D structures from in vivo tissue and in vitro basement matrix embedded organoids, and detail different fixation and staining protocols optimized for immuno-labeling of microtubule, centrosomal, and junctional proteins as well as cell markers including the stem cell protein Lgr5.

The advent of 3D in vitro organoids that mimic the in vivo tissue architecture and morphogenesis has greatly advanced the ability to study key biological ...

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

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

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

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

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 ...

Genetic Engineering of Primary Mouse Intestinal Organoids Using Magnetic Nanoparticle Transduction Viral Vectors for Frozen Sectioning

Intestinal organoid cultures provide a unique opportunity to investigate intestinal stem cell and crypt biology in vitro, although efficient approaches to manipulate gene expression in organoids have made limited progress in this arena. While CRISPR/Cas9 technology allows for precise genome editing of cells for organoid generation, this strategy requires extensive selection and screening by sequence analysis, which is both time-consuming and costly. Here, we provide a detailed protocol for efficient viral transduction of intestinal organoids. This approach is rapid and highly efficient, thus decreasing the time and expense inherent in CRISPR/Cas9 technology. We also present a protocol to generate frozen sections from intact organoid cultures for further analysis with immunohistochemical or immunofluorescent staining, which can be used to confirm gene expression or silencing. After successful transduction of viral vectors for gene expression or silencing is achieved, intestinal stem cell and crypt function can be rapidly assessed. Although most organoid studies employ in vitro assays, organoids can also be delivered to mice for in vivo functional analyses. Moreover, our approaches are advantageous for predicting therapeutic responses to drugs because currently available therapies generally function by modulating gene expression or protein function rather than altering the genome.

We describe step-by-step instructions to: 1) efficiently engineer intestinal organoids using magnetic nanoparticles for lenti- or retroviral transduction, and, 2) generate frozen sections from engineered organoids. This approach provides a powerful tool to efficiently alter gene expression in organoids for investigation of downstream effects.

Intestinal organoid cultures provide a unique opportunity to investigate intestinal stem cell and crypt biology in vitro, although efficient approaches to ...

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 ...

Generation of Multicellular Human Primary Endometrial Organoids

The human endometrium is one of the most hormonally responsive tissues in the body and is essential for the establishment of pregnancy. This tissue can also become diseased and cause morbidity and even death. Model systems to study human endometrial biology have been limited to in vitro culture systems of single cell types. In addition, the epithelial cells, one of the major cell types of the endometrium, do not propagate well or retain their physiological traits in culture, and thus our understanding of endometrial biology remains limited. We have generated, for the first time, endometrial organoids that consist of both epithelial and stromal cells of the human endometrium. These organoids do not require any exogenous scaffold materials and specifically organize so that epithelial cells encompass the spheroid-like structure and become polarized with stromal cells in the center that produce and secrete collagen. Estrogen, progesterone and androgen receptors are expressed in the epithelial and stromal cells and treatment with physiological levels of estrogen and testosterone promote the organization of the organoids. This new model system can be used to study normal endometrial biology and disease in ways that were not possible before.

A protocol to generate human primary endometrial organoids that consist of epithelial and stromal cells and retain characteristics of the native endometrial tissue is presented. This protocol describes methods from uterine tissue acquisition to the histologic processing of endometrial organoids.

The human endometrium is one of the most hormonally responsive tissues in the body and is essential for the establishment of pregnancy. This tissue can ...

Recapitulating Suckling-to-Weaning Transition In Vitro using Fetal Intestinal Organoids

At the end of the suckling period, many mammalian species undergo major changes in the intestinal epithelium that are associated with the capability to digest solid food. This process is termed suckling-to-weaning transition and results in the replacement of neonatal epithelium with adult epithelium which goes hand in hand with metabolic and morphological adjustments. These complex developmental changes are the result of a genetic program that is intrinsic to the intestinal epithelial cells but can, to some extent, be modulated by extrinsic factors. Prolonged culture of mouse primary intestinal epithelial cells from late fetal period, recapitulates suckling-to-weaning transition in vitro. Here, we describe a detailed protocol for mouse fetal intestinal organoid culture best suited to model this process in vitro. We describe several useful assays designed to monitor the change of intestinal functions associated with suckling-to-weaning transition over time. Additionally, we include an example of an extrinsic factor that is capable to affect suckling-to-weaning transition in vivo, as a representation of modulating the timing of suckling-to-weaning transition in vitro. This in vitro approach can be used to study molecular mechanisms of the suckling-to-weaning transition as well as modulators of this process. Importantly, with respect to animal ethics in research, replacing in vivo models by this in vitro model contributes to refinement of animal experiments and possibly to a reduction in the use of animals to study gut maturation processes.

This protocol describes how to mimic suckling-to-weaning transition in vitro using mouse late fetal intestinal organoids cultured for 30 days.

At the end of the suckling period, many mammalian species undergo major changes in the intestinal epithelium that are associated with the capability to ...

Generation of Mosaic Mammary Organoids by Differential Trypsinization

Organoids offer self-organizing, three-dimensional tissue structures that recapitulate physiological processes in the convenience of a dish. The murine mammary gland is composed of two distinct epithelial cell compartments, serving different functions: the outer, contractile myoepithelial compartment and the inner, secretory luminal compartment. Here, we describe a method by which the cells comprising these compartments are isolated and then combined to investigate their individual lineage contributions to mammary gland morphogenesis and differentiation. The method is simple and efficient and does not require sophisticated separation technologies such as fluorescence activated cell sorting. Instead, we harvest and enzymatically digest the tissue, seed the epithelium on adherent tissue culture dishes, and then use differential trypsinization to separate myoepithelial from luminal cells with ~90% purity. The cells are then plated in an extracellular matrix where they organize into bilayered, three-dimensional (3D) organoids that can be differentiated to produce milk after 10 days in culture. To test the effects of genetic mutations, cells can be harvested from wild type or genetically engineered mouse models, or they can be genetically manipulated prior to 3D culture. This technique can be used to generate mosaic organoids that allow investigation of gene function specifically in the luminal or myoepithelial compartment.

The mammary gland is a bilayered structure, comprising outer myoepithelial and inner luminal epithelial cells. Presented is a protocol to prepare organoids using differential trypsinization. This efficient method allows researchers to separately manipulate these two cell types to explore questions concerning their roles in mammary gland form and function.

Organoids offer self-organizing, three-dimensional tissue structures that recapitulate physiological processes in the convenience of a dish. The murine ...

A Simplified Method for Generating Kidney Organoids from Human Pluripotent Stem Cells

Kidney organoids generated from hPSCs have provided an unlimited source of renal tissue. Human kidney organoids are an invaluable tool for studying kidney disease and injury, developing cell-based therapies, and testing new therapeutics. For such applications, large numbers of uniform organoids and highly reproducible assays are needed. We have built upon our previously published kidney organoid protocol to improve the overall health of the organoids. This simple, robust 3D protocol involves the formation of uniform embryoid bodies in minimum component medium containing lipids, insulin-transferrin-selenium-ethanolamine supplement and polyvinyl alcohol with GSK3 inhibitor (CHIR99021) for 3 days, followed by culture in knock-out serum replacement (KOSR)-containing medium. In addition, agitating assays allows for reduction in clumping of the embryoid bodies and maintaining a uniform size, which is important for reducing variability between organoids. Overall, the protocol provides a fast, efficient, and cost-effective method for generating large quantities of kidney organoids.

Here we describe a protocol to generate kidney organoids from human pluripotent stem cells (hPSCs). This protocol generates kidney organoids within two weeks. The resulting kidney organoids can be cultured in large-scale spinner flasks or multi-well magnetic stir plates for parallel drug-testing approaches.

Kidney organoids generated from hPSCs have provided an unlimited source of renal tissue. Human kidney organoids are an invaluable tool for studying kidney ...

Innervation of Human Intestinal Organoids

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Generation of iPSC-derived Human Brain Organoids to Model Early Neurodevelopmental Disorders

Immuno-fluorescent Labeling of Microtubules and Centrosomal Proteins in Ex Vivo Intestinal Tissue and 3D In Vitro Intestinal Organoids

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

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

Genetic Engineering of Primary Mouse Intestinal Organoids Using Magnetic Nanoparticle Transduction Viral Vectors for Frozen Sectioning

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

Generation of Multicellular Human Primary Endometrial Organoids

Recapitulating Suckling-to-Weaning Transition In Vitro using Fetal Intestinal Organoids

Generation of Mosaic Mammary Organoids by Differential Trypsinization

A Simplified Method for Generating Kidney Organoids from Human Pluripotent Stem Cells