Innervation of Human Intestinal Organoids

Summary

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.

Abstract

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 >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.

Introduction

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 mortality^1,2,3,4. 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) lines^6,7. Their protocol included a carefully timed series of incubations with specific growth factors to mimic human fetal intestinal development. Activin A, a TGFβ 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 fate^8,9. 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's HIO + ENS with greater neuronal subtype diversity and neuroepithelial synaptic connections not seen in HIO + ENS. Importantly, the ENCCs used in Schlieve'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'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'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'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.

Protocol

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 hESCs^9,10,12, H1 hESCs⁹, hESC line UCSF4¹², and multiple hiPSC lines such as WTC11¹², WTC11 AAVS1-CAG-GCaMP6f⁹, WTC10^9,10, WTC10 PHOX2B het (+/Y14X)⁹, and WTC10 PHOX2B null (Y14X/Y14X)⁹.

1. 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 °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 °C. Store with adequate diluent to cover the entire well to prevent the coating from drying out.
2. 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 °C, 5% CO₂, and change the media every other day with 2 mL of stem cell media per well.
3. 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 °C.
   NOTE: Waiting until cells are past 80% confluency to passage may cause problems with premature differentiation, which will reduce yield on subsequent steps.
4. 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.
5. 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 µL 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.

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 "Enteric Neural Crest (ENC) Induction (Days 0-12)" and "ENCC Spheroid Formation (Day 12-15)" in Barber et al.^10,12.

1. 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 µL of hPSC cell suspension from step 1.5 to the same well (typically a 5:6 passaging ratio).
2. Maintain cells at 37 °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.
3. ENC induction
   1. 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.
   2. 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.
   3. 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.
   4. 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.
4. ENCC spheroid formation
   1. 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 °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.
   2. Centrifuge the cell suspension in the 15 mL conical tube at 300 × g for 1 min. Aspirate the supernatant. Gently resuspend the pellet in 2 mL of warm NCC media.
   3. Add the resuspended cells to a new, uncoated well of an ultra-low-binding plate and incubate at 37 °C, 5% CO₂ for 48 h.
   4. 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.

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 labs^6,7. The process occurs in two phases: Definitive endoderm (DE) induction and Mid-hindgut spheroid formation.

1. Preparation for HIO Generation
   1. 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 µL per well and incubate at 37 °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 °C. Store with enough diluent to cover the entire well to prevent the coating from drying out.
      ​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.
   2. 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 µL into each well (e.g., if 850 µL of hPSC cell suspension is left after passaging the hPSC maintenance plate, then for 6 wells of a 24-well plate, mix 2,150 µL of warm stem cell media with 850 µL of the hPSC cell suspension from step 1.5, and 500 µL distributed in each of 6 wells).
      NOTE: As we typically passage hPSC maintenance plates with volumes of 50-150 µL, we usually have volumes of 850-950 µL available for this step. Thus, the volume of hPSCs per well in the 24-well plate approximates 150 µL/well.
      ​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.
   3. Maintain cells at 37 °C, 5% CO₂. Change the media every other day with 500 µL 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 >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.
2. DE induction
   1. Day 1. Once hPSCs have reached 50-70% confluency, aspirate the stem cell media from each well completely and replace with 500 µL of warm Day 1 DE media (Table 1) per well. Incubate at 37 °C, 5% CO₂ for 24 h.
   2. Day 2. Aspirate the Day 1 DE and replace with 500 µL of warm Day 2 DE (Table 1) media per well. Incubate at 37 °C, 5% CO₂ for 24 h.
   3. Day 3. Aspirate the Day 2 DE and replace with 500 µL of warm Day 3 DE (Table 1) media per well. Incubate at 37 °C, 5% CO₂ for 24 h.
3. Mid-hindgut spheroid formation
   1. Days 4-8. Aspirate the media from each well completely and replace with 500 µL of warm Mid-hindgut media (MHGM, Table 1) per well. Incubate at 37 °C, 5% CO₂. Repeat this step daily until Day 8, at which time mid-hindgut spheroids are ready for collection and coculture (Section 4).

4. Co-culture of mid-hindgut spheroids with ENCCs

NOTE: A graphic summary of this section is provided in Figure 1.

1. Preparation of ENCC suspension
   1. 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.
   2. Add 1 mL of a warm enzymatic cell detachment reagent to the well and incubate at 37 °C for 30 min.
   3. 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.
   4. 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.
   5. Centrifuge the cell suspension at 300 × g for 1 min. Aspirate the supernatant with a sterile pipette tip.
   6. 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.
2. Combining ENCC suspension with mid-hindgut spheroids
   1. 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.
   2. 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.
   3. 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.
   4. Centrifuge at 300 × 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 µL per final coculture well.
      NOTE: Avoid forming bubbles in the clear basement 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.
   5. To the center of each dry well, pipette a 30 µL 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 °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 membrane matrix.
3. In Vitro HIO+ENS coculture
   1. Day 0: Once the clear basement membrane matrix has polymerized, cover each droplet in 500 µL of warm Day 0-3 HIO media (see Table 1).
   2. 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 µL 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.
   3. Days 7-14: Continue replacing Day 3-14 HIO media as needed every 3-4 days and monitor the growth.
4. Splitting HIOs
   1. 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.
   2. Prepare 2 mL microcentrifuge tubes with 30 µL of clear basement membrane matrix and keep these on ice.
   3. 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.
   4. Place each isolated HIO + ENS into one of the prepared microcentrifuge tubes containing 30 µL 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.
   5. Repeat step 4.2.5 for the split and replated HIO+ENS. Once the clear 3D basement membrane droplets are polymerized, add 500 µL of warm, fresh Day 3-14 HIO media to each well and incubate overnight.
5. HIO+ENS coculture continued
   1. Day 15: Carefully aspirate the Day 3-14 HIO media from the side of the well. Replace the media with 500 µL warm Day 15-28 HIO media (see Table 1).
   2. Day 15-40: Continue replacing Day 15-28 HIO media as needed every 3-4 days and monitoring HIO + ENCC growth.

Results

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, differen...

Discussion

HIOs have been used as a model system for human intestinal development since the early 2010'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 spher...

Materials

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