Apr 15, 2025
JAX_DPC: Protocol for definitive endoderm induction of human induced pluripotent stem cells
- 1The Jackson Laboratory for Genomic Medicine;
- 2UCONN Health
- MorPhiC Consortium
Protocol Citation: Cole Lorig, Juliana Alcoforado Diniz, Paul Robson 2025. JAX_DPC: Protocol for definitive endoderm induction of human induced pluripotent stem cells. protocols.io https://dx.doi.org/10.17504/protocols.io.14egn4zrpv5d/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: April 11, 2025
Last Modified: April 15, 2025
Protocol Integer ID: 126551
Funders Acknowledgements:
JAX MorPhiC DPC
Grant ID: 1 UM1 HG012651-01
Abstract
The definitive endoderm forms as the bottom layer of trilaminar embryonic disc and develops the innermost germ layer of the embryo. It is the progenitor of the epithelial lining that further differentiates into organs of both the digestive tract and respiratory system. Studying endoderm models offers opportunity to investigate early embryonic development, cell fate mapping, and lineage analysis. This monolayer protocol is adapted from the STEMdiffTM Definitive Endoderm Kit and describes a method for induction of definitive endoderm from human induced pluripotent stem cells (hiPSCs).
Materials
MATERIALS
mTeSR plus (STEMCELL technologies)
KnockOut Serum Replacement (ThermoFisher Scientific, 10828028)
KnockOut DMEM (Thermo
Fisher Scientific, 10829018)
Neurobasal Medium (Gibco, 21103049)
L-glutamine (Gibco, 25030081)
MEM non-essential amino acids (NEAA) (Gibco),
penicillin-streptomycin (Gibco),
b-mercaptoethanol (Gibco, 21985023)
N2 supplement (100X) (ThermoFisher, 17502048)
B27 supplement without vitamin A 5OX (ThermoFisher, 12587010)
D-(+)-Glucose solution (Sigma, G8644)
Glutamax (100x) (Gibco, 35050-061)
HEPES pH7.4 (Fisher, 15630080)
BDNF (R&D Systems, 248-BDB-010)
GDNF (R&D Systems, 212-GD-010)
Accutase (StemCell Technologies, 7922)
Reagents and Consumables:
Reagents and Consumables:
STEMdiffTM Definitive Endoderm Kit (STEMCELL Technologies, #05110)
- STEMdiffTM Endoderm Basal Medium (STEMCELL Technologies, #05111)
- STEMdiffTM Definitive Endoderm Supplement MR (100X) (STEMCELL Technologies, #05112)
- STEMdiffTM Definitive Endoderm Supplement CJ (20X) (STEMCELL Technologies, #05113)
mTeSRTM Plus (STEMCELL Technologies, #100-0276)
Corning‱ Matrigel‱ Basement Membrane Matrix, LDEV-free (Corning, 354234)
DMEM/F-12 with 15 mM HEPES (STEMCELL Technologies, #36254)
GibcoTM DPBS, no calcium, no magnesium (ThermoFisher, 14190144)
Gentle Cell Dissociation Reagent (STEMCELL Technologies, #100-0485)
Y-27632 (dihydrochloride) (STEMCELL Technologies, #72304)
GibcoTM Trypan Blue 0.4% (ThermoFisher, 15250061)
Corning‱ Costar‱ TC-Treated 6-Well Plates (Millipore-Sigma, CLS3516)
Procedure:
Procedure:
Maintain iPSCs in a Matrigel coated 6-well plate with 2 mL of mTeSR Plus media.
Perform daily media change until the well is approximately 70-80% confluent for Day 0.
Day 0
Coat 6-well plate in Matrigel
a. Resuspend a 100 uL aliquot of Matrigel in 6 mL of DMEM/F-12 and add 1 mL to each well of a 6-well plate.
b. Place plate in a 37°C incubator for at least 2 hours
Warm sufficient volumes of mTeSR plus and DMEM/F-12 to 37°C
Prepare plating media by adding Y-27632 to mTeSR Plus for a final concentration of 10 µM
a. Add 2 uL of 5 mM Y-27632 for every 1 mL of mTeSR
Taking the plate of iPSCs, wash the well with 1 mL of D-PBS
Aspirate the wash and add 1 mL of Gentle Cell Dissociation Reagent
Incubate plate at 37°C for 10 minutes
While cells are incubating
a. Remove the Matrigel coated plate (from Step 1) from the incubator. Aspirate the Matrigel and wash each well with 1 mL of DMEM/F-12
b. Add 2 mL of plating medium (mTeSR + Y-27632)
c. Place plate back into incubator until ready to seed cells
After 10 minutes, triturate cells to create a single cell suspension and transfer the cells to a 15 mL conical tube containing 1 mL of mTeSR. Rinse well with an additional 1 mL of mTeSR and transfer to the same conical tube
Centrifuge cells at 300 x g for 5 minutes
Carefully aspirate out supernatant and resuspend cell pellet in 1 mL of plating medium (mTeSR + Y-27632).
Count cells by mixing 10 uL of cell suspension with 10 uL of Trypan Blue and count using hemocytometer
Plate cells at a density of 1x106 cells per well to the prepared plate (Step 7).
a. Cells should be 90-100% confluent on Day 1
Incubate cells at 37°C for 24 Hours
Day 1
Warm sufficient volumes of DMEM/F-12 and STEMdiffTM Definitive Endoderm Basal Medium for Day 1 use
Prepare Medium 1 as follows:
a. Thaw STEMdiffTM Definitive Endoderm Supplement MR and STEMdiffTM Definitive Endoderm Supplement CJ on ice
b. Dilute both supplements 1 in 100 in STEMdiff‱ Endoderm Basal Medium
Aspirate medium from the well and wash with 1 mL of DMEM/F-12
Aspirate DMEM/F-12 and replace with 2 mL of Medium 1
Incubate at 37°C for 24 hours
Day 2
Prepare Medium 2. Prepare sufficient Medium 2 to be used on Days 2, 3, and 4
a. Thaw STEMdiff‱ Definitive Endoderm Supplement CJ on ice
b. Add Supplement CJ to cold (2 - 8°C) STEMdiff‱ Endoderm Basal Medium at a 1 in 100 dilution
c. Store media for days 3 and 4 media at 2 - 8°C
Warm sufficient volume of Medium 2 necessary for Day 2 (2 mL per well) to 37°C
Taking the plate from the incubator, aspirate medium and add 2 mL of Medium 2
Incubate at 37°C for 24 hours
Day 3
Warm sufficient volume of Medium 2 necessary for Day 3 (2 mL per well) to 37°C
Taking the plate from the incubator, aspirate medium and add 2 mL of Medium 2
Incubate at 37°C for 24 hours
Day 4
Warm sufficient volume of Medium 2 necessary for Day 4 (2 mL per well) to 37°C
Taking the plate from the incubator, aspirate medium and add 2 mL of Medium 2
Incubate at 37°C for 24 hours
Day 5
Cells are ready to be assayed for the formation of definitive endoderm