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

Summary

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

Abstract

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

Introduction

Studying human colon development is difficult due to restrictions on the use of human fetal tissue. Animal models have been invaluable and historically used for genetic approaches in mice to study intestinal development. However, differences between mouse and human intestinal development limit the applicability of mice as a model system. For instance, although crypt formation in the small intestine and colon of mice occurs postnatally, humans are born with fully formed crypts¹. Furthermore, the human small intestine and colon contain cell types that are not found in mice, including motilin (MLN)-expressing enteroendocrine cells in the small int....

Protocol

1. Generation of human intestinal and colonic organoids

1. Preparing ECM-coated plates
   1. Add 50 mL of cold DMEM medium into a 50 mL conical tube.
   2. Remove an aliquot of 4x hESC-qualified ECM (see the Table of Materials) from the -80 °C freezer and thaw on ice.
      NOTE: Refer to the product's certificate of analysis to determine the volume of ESC-qualified ECM required to prepare a 4x stock.
   3. If hESC-qualified ECM is not fully thawed, take 750 µL of DMEM from the 50 mL conical tube and mix it with the ECM.
   4. Transfer the DMEM/hESC-qualified ECM mixture to the 50 mL conical tube with DMEM....

Results

The successful generation of spheroids during the mid/hindgut induction stage is indicative of successful patterning. Perform IF staining for CDX2 on floating spheroids and on the monolayer to confirm that patterning is correct. Although staining at the definitive endoderm (DE) stage can indicate the effectiveness of DE induction, spheroid generation is not possible without efficient DE induction. To test the efficiency of DE induction, perform IF staining and/or RT-qPCR for FOXA2 and SOX17.

F.......

Discussion

The differentiation of hPSCs into HIOs and HCOs is a complex process requiring quality controls at each step. The starting hPSCs need to have minimal differentiation before initiating differentiation into DE. Optimizing the density of hPSCs plated for DE differentiation is critical for the success of the protocol. To ensure the quality of DE differentiation, perform IF for FOXA2 and SOX17 to determine the efficiency of DE differentiation. DE differentiation should result in over 80% of the treated cells staining positive.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1% Bovine serum albumin (BSA) solution	N/A	N/A	N/A
15 mL Corning tube	Falcon	21008-918	N/A
30% Sucrose	N/A	N/A	Made in PBS.
5% Normal donkey serum	Jackson ImmunoResearch Lab	017-000-121	N/A
50 mL Corning tube	Falcon	21008-951	N/A
Accutase	Thermo Scientific	A1110501	Cell detachment solution; aliquot 5 mL of Accutase into 10 mL tubes totaling 20 tubes and store at -20 °C for up to 6 months. Place at 4 °C overnight before use.
Activin A	Cell guidance Systems	GFH6-100x10	Reconstitute the lyophilized powder at 100 µg/mL in sterile PBS containing 0.1% bovine serum albumin (BSA). Aliquot 38 µL of Activin A into prechilled microcentrifuge tubes and store at -80 °C (Tubes expire 12 months from date of receipt).
Activin Day 1 medium (RPMI 1640)	Corning	MT10041CV	Use nonessential amino acids (NEAA, Corning 11140050) and store at 4 °C. Basic day 1 medium: 500 mL of RPMI 1640 and 500 mL of NEAA. When preparing Activin Day 1 medium, add 13 mL of basic day 1 medium, 13 µl of Activin A (100 µg/mL), and 2 µl of BMP4 (100 µg/mL). The base medium is stable for up to 3 weeks but should be used immediately after addition of growth factors.
Activin Day 2 medium (RPMI 1640, 0.2% FBS vol/vol)	Hyclone	SH30070.03T	Use nonessential amino acids (Corning 11140050) and store at 4 °C. Basic day 2 medium: 500 mL of RPMI 1640, 500 mL of NEAA, and 1 mL of 0.2% serum. When preparing Activin Day 2 medium, add 12.5 mL of basic day 2 medium and 12.5 µL of Activin A (100 µg/mL). The base medium is stable for up to 3 weeks but should be used immediately after addition of growth factors.
Activin Day 3 medium (RPMI 1640, 2% FBS vol/vol)	Hyclone	SH30070.03T	Use nonessential amino acids (Corning 11140050) and store at 4 °C. Basic day 3 medium: 500 mL of RPMI 1640, 500 mL NEAA, and 10 mL of 2% serum. When preparing Activin Day 3 medium, add 12.5 mL of basic day 3 and 12.5 µL of Activin A (100 µg/mL). The base medium is stable for up to 3 weeks but should be used immediately after addition of growth factors.
Alexa Fluor 488 Donkey anti-Goat	Thermo Scientific	A11055	1:500 dilution (Secondary antibody)
Alexa Fluor 488 Donkey anti-Rabbit	Thermo Scientific	A21206	1:500 dilution (Secondary antibody)
Alexa Fluor 546 Donkey anti-Mouse	Thermo Scientific	A10036	1:500 dilution (Secondary antibody)
Alexa Fluor 647 Donkey anti-Mouse	Thermo Scientific	A31571	1:500 dilution (Secondary antibody)
Base mold	Fisher	22-363-552	N/A
Basic gut medium (advanced DMEM)	Gibco	12491015	When preparing Basic gut medium, add 500 mL of DMEM, 500 mL of N2 (Gibco 17-502-048), 500 mL of B27 (Gibco), 500 mL of L-Glutamine to get 2 mM L-Glutamine (Corning A2916801), 5 mL of 100 U/mL Penicillin-Streptomycin (Gibco 15-140-122), and 7.5 mL of  1 M HEPES to get 15 mM HEPES.  The base medium is stable for up to 3 weeks but should be used immediately after addition of growth factors.
Biorad CFX96 Touch Real-Time PCR Detection System	Biorad	N/A	Other qRT-PCR systems can be used.
Cell Recovery Solution	Corning	354253	ECM-degrading solution
CHIR99021	Reprocell	4000410	Reconstitute by adding 2.15 mL of DMSO at 10 mM. Prepare 50 µL aliquots and store at -20 °C.  Store powder at 4 °C, protected from light.
CTRL HIO patterning medium	N/A	N/A	Basic gut medium and 100 ng/mL EGF.
DAPI	Sigma-Aldrich	D9542	1:100 dilution (Secondary antibody)
DE monolayer	N/A	N/A	Monolayer was generated in prior steps (Section 4.4).
Dispase	Gibco	17105041	Resuspend lyophilized powder in Advanced DMEM (Gibco MT15090CV) to a 1 mg/mL final concentration. Filter the solution for sterilization by vacuuming using a Millipore filter sterilization tube. Make 10 mL aliquots (1 mg/mL) and store at -20 °C for up to 6 months. Place at 4 °C overnight before use.
EGF	Thermo Scientific	236-EG-01M	When preparing 100 ng/mL EGF reconstitute 500 µg/mL in sterile PBS. Next add 2 mL of sterile PBS to 1 mg EGF and make 500 µg/mL EGF solution. Aliquot 100 µL of EGF in 20 tubes.
Fisherbrand 6 cm Petri Dishes with Clear Lid	Fisher	FB0875713A	N/A
Fisherbrand Cell Lifter	Fisher	08-100-240	N/A
Fisherbrand Class B Clear Glass Threaded Vials with Closures Attached	Fisher	03-338B	N/A
Fisherbrand Disposable Borosilicate Glass Pasteur Pipette	Fisher	13-678-2D0	N/A
Fluoromount G Slide Mounting Medium	VWR	100241-874	N/A
Gibco advanced DMEM	Gibco	12-491-023	N/A
Goat anti-E-Cadherin	R&D systems	AF648	1:400 dilution (Primary antibody)
Goat anti-SOX17	R&D systems	AF1924	1:500 dilution (Primary antibody)
HCOs patterning medium	N/A	N/A	Basic gut medium, 100 ng/mL EGF and 100 ng/mL BMP2. When preparing BMP2, add 1 mL of sterile 4 mM HCl 0.1% BSA to BMP2 vials (100 µg). Aliquot 25 µL of BMP4 solution in 4 tubes.  The base medium is stable for up to 3 weeks but should be used immediately after addition of growth factors.
Hemocytometer	Sigma-Aldrich	Z359629	N/A
Human Pluripotent Stem Cells (hPSC)	Pluripotent Stem Cell Facility	N/A	Cells seeded in a Matrigel coated 24-well plate (Thermo Scientific 73520-906).
Ice-cold 4% Paraformaldehyde solution (PFA)	N/A	N/A	N/A
Ice-cold Phosphate Buffered Saline (PBS)	N/A	N/A	The pH must be 7.4.
ImmEdge Hydrophobic Barrier Pen	Vector Laboratories	101098-065	N/A
Induced Pluripotent Stem Cells (iPSCs)	Pluripotent Stem Cell Facility (Cincinnati Children's Hospital Medical Center)	N/A	Other hESC or iPSC lines can be used, but the protocol needs to be optimized for each cell line.
Leica microtome	N/A	N/A	N/A
LSM 880			confocal microscope
Matrigel Basement Membrane Matrix	Corning	354234	N/A
Matrigel hESC-qualified Matrix	Corning	354277	Prepare 4 x Matrigel aliquots which corresponds to volumes sufficient to make enough diluted Matrigel for 4 x 6-well dishes.
Mid-hindgut induction medium (RPMI 1640)	Corning	MT10041CV	Nonessential amino acids (Corning 11140050), 2% FBS vol/vol (Hyclone SH30070.03T), 3 µM CHIR99021 and 500 ng/mL FGF4. The base medium is stable for up to 3 weeks but should be used immediately after addition of growth factors.
Mid-hindgut spheroids	N/A	N/A	N/A
MilliporeSigma Steriflip Sterile Disposable Vacuum Filter Units	MilliporeSigma	SCGP00525	N/A
Mouse anti-CDX2	BioGenex	MU392-UC	1:300 dilution (Primary antibody)
Mouse anti-FOXA2	Abnova/Novus	H00003170-M01	1:500 dilution
mTeSR1 complete growth medium	Stem Cell technologies	85870	Add 100-mL of mTeSR supplement (85870) into one 400-mL mTeSR medium (85870) and aliquot into 50-mL tubes while avoiding contamination. Store at 4°C until use.
Murray's Clear solution (Also known as BABB)	Murray's	N/A	1:2 benzyl benzoate and benzyl alcohol.
NOG HIO patterning medium	N/A	N/A	Basic gut medium, 100 ng/mL EGF and 100 ng/mL NOGGIN (Dispense 25 µg of NOGGIN in 250 µl sterile PBS with 0.1% BSA).
NucleoSpin RNA	Takara	740955.25	Other RNA isolation kits may be used.
Nunclon delta surface tissue culture dish 24-wells (Nunc)	Thermo Scientific	73521-004	N/A
Nunclon delta surface tissue culture dish 24-wells coated with Matrigel	Thermo Scientific	73521-004	N/A
Nunclon delta surface tissue culture dish 6-wells (Nunc)	Thermo Scientific	73520-906	N/A
Nunclon delta surface tissue culture dish 6-wells coated with  Matrigel.	Thermo Scientific	73520-906	N/A
Outgrowth medium for HIOs, CTRL HIOs, and HCOs	N/A	N/A	Basic gut medium and 100 ng/mL EGF (Final concentration)
Phosphate Buffer Saline, 0.5% Triton X (PBS-T)	N/A	N/A	N/A
Primers	Integrated DNA Technologies, Inc. (IDT)	N/A	The primers are listed in Table 2 on the protocol.
Rabbit anti-CDX2	Cell Marque	EPR22764Y	1:100 dilution (Primary antibody)
Rabbit anti-SATB2	Cell Marque	EP281	1:100 dilution (Primary antibody)
Recombinant Human BMP-4 Protein	R&D systems	314-BP-010	Reconstitute the lyophilized powder at 100 µg/mL in sterile 4 mM HCl containing 0.1% bovine serum albumin (BSA). Add 4.17 mL HCl solution to 45.83 mL molecular water totaling to 50 mL of 1 M HCl. Then add 200 µL of 1 M HCl to 49.8 mL of molecular grade water totaling to 50 mL of 4 mM HCl. Next add 0.05 g BSA to 50 mL of 4 mM HCl and filter to make sterile. Aliquot sterile 4 mM HCl 0.1% BSA to 33 microcentrifuge tube totaling and store at -20 °C. Add 100 µl of sterile 4 mM HCl 0.1% BSA to the BMP4 vials (10 µg) to make BMP4 solution at 100 µg/mL.
Recombinant Human FGF-4 Protein	R&D systems	235-F4-01M	Reconstitute at 100 µg/mL in sterile PBS containing 0.1% bovine serum albumin. Add 0.05 g of BSA in 50 mL of PBS to make 0.1% BSA. Filter 0.22 µM BSA to sterilize the BSA. Aliquot 10 mL of 0.1% BSA in 5 tubes. Add 1 mg FGF-4 in 10 mL of sterile 0.1% BSA. Aliquot 250 µL into prechilled 40 microcentrifuge tubes and store at -80 °C.
ROCK inhibitor Y-27632	Tocris	1254	The final concentration is 10 mM (10 mmol/L). Resuspend in DMSO at 10 mM and filter sterilize. Add 3 mL of sterile PBS to each vial. Aliqout 100 µL of ROCK inhibitor in 30 tubes and store at -20 °C.
SuperScript VILO cDNA Synthesis Kit	Thermo Scientific	11-754-250	N/A
SuperFrost Plus microscope slides
Tissue Tek O.C.T Compound	VWR	25608-930	N/A