Jun 19, 2024
Human RA-gastruloid induction from pluripotent stem cells
- Nobuhiko Hamazaki1
- 1University of Washington
Protocol Citation: Nobuhiko Hamazaki 2024. Human RA-gastruloid induction from pluripotent stem cells. protocols.io https://dx.doi.org/10.17504/protocols.io.261ge5epog47/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: June 17, 2024
Last Modified: June 19, 2024
Protocol Integer ID: 101954
Keywords: Embryo model, Stem cell, Gastruloid
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Abstract
Gastruloids are a powerful in vitro model of early human development. However, although elongated and composed of all three germ layers, human gastruloids do not morphologically resemble post-implantation human embryos. Here we show an early pulse of retinoic acid (RA), together with Matrigel, robustly induces human gastruloids with posterior embryo-like morphological structures, including a neural tube flanked by segmented somites, and diverse cell types including neural crest, neural progenitors, renal progenitors, and myocytes. Through in silico staging based on single-cell RNA-seq (scRNA-seq), we find human RA-gastruloids to be more advanced than other embryo models, and comparable to E9.5 mouse and CS11 cynomolgus monkey embryos. We leverage chemical and genetic perturbations of RA-gastruloids to confirm that WNT and BMP signalling regulate somite formation and neural tube length in the human context, while transcription factors TBX6 and PAX3 underpin presomitic mesoderm and neural crest, respectively. Looking forward, RA-gastruloids are a robust, scalable model for decoding early human embryogenesis.
Materials
- StemPro Accutase (Thermo, A1110501)
- Wash media
Note
DMEM/F-12 (Thermo, 11320033) + 0.1% BSA (Thermo, 15260037)
- 10 µM Y-27632 (Sellek, S1049)
- Nutristem hPSC XF medium (Biological Industries, 05-100-1A)
- 30mM CHIR99021 (CHIR, Millipore, SML1046)
Note
(Make 6µL aliquots and stored in -20C, and test the batch before the use)
- 100mM Retinoic Acid (RA) stock (Millipore Sigma, R2625)
Note
Make 10µL aliquots and stored in -20C)
- Essential 6 medium (Thermo, A1516401)
- Matrigel (Corning, 354230)
- Non-adhesive 96-well plate, such as Nunclon‱ Sphera‱ 96-Well, Nunclon Sphera-Treated,U-Shaped-Bottom Microplate and PrimeSurface‱ 3D culture: Ultra-low Attachment Plates: 96 well,U bottom, Clear plates
- Vitronectin (Gibco, A14700)
Note
Make 200 µl aliquots and store them in -20C and dilute them x100 in PBS before the use and keep remaining in 4C and use it within 2 weeks
Day0, Passage human PSCs onto vitronectin-coated NutriStem
Day0, Passage human PSCs onto vitronectin-coated NutriStem
Coat wells with Vitronectin and incubate them in 37C for at least 15 min
Dissociate pre-treated ESCs
Aspirate media from wells and wash wells with PBS(-) and aspirate PBS (-)
Add 500 µL Accutase to each well and incubate them at 37C for 4 min
Quench the reaction by adding 2 mL of Wash media containing 10 µM Y-27632 media and pipette up and down ~10 times in a well to dissociate cells
Transfer them to a new 15mL tube and centrifuge for 3 min at 300 g
Suspend them in NutriStem containing 10 µM Y-27632 and spread 2 × 104 cells for 1/12 well onto each vitronectin-coated well
Note
The number of seeding cells on the pre-treatment plate is important for later gastruloid elongation and needs to be optimized for each lab and each cell line.
Day 1-3, Pre-treatment of ESCs (CHIR stimulation)
Day 1-3, Pre-treatment of ESCs (CHIR stimulation)
On day 1, Change medium to fresh NutriStem medium containing 5 µM Y-27632
On day 2, Change the medium to fresh NutriStem medium containing 4 µM CHIR
Note
- CHIR concentrations and duration of treatment should be optimized for each CHIR batch and/or each cell line.
- CHIR should be used within three months from the aliquoting.
On day 3, Change medium to fresh NutriStem medium containing 4 µM CHIR + 500 nM RA
Note
- The concentrations of CHIR and RA should be optimized for each cell line.
Day 4-9, Human RA-gastruloid induction
Day 4-9, Human RA-gastruloid induction
On day 4, prepare the following reagents
Note
- Accutase = 500µL for 1/12 well
- Wash media + 10 µM Y-27632 = 2 mL for 1/12 well
- Essential 6 medium + 1 µM CHIR + 5 µM Y-27632 = 50µL / well + 10%
Aspirate media from wells and wash wells with PBS(-) and aspirate PBS (-)
Add 500µL Accutase to each well and incubate them in 37C for 4 min
Quench the reaction by adding 2 mL of Wash+Y media to each well and pipette up and down ~10 times in a well to dissociate cells
Transfer them to a new 15 mL tube and centrifuge for 3 min at 300 g
Wash again with 2 mL Essential6 + 1µM CHIR + Y and centrifuge it again
- Resuspend them in 1-2 mL Essential 6 + Y medium and count cells by Countess
Transfer the necessary number of cells (4000 cells/well) into a reservoir containing a medium (50 µl/well)
Spread cells with a multi-channel pipette
Incubate them in 37C incubator
- On day 5 (+24h from RA-gastruloid induction), add 150 µL Essential 6 media to each well.
- On day 6 (+48h from RA-gastruloid induction), carefully remove 150 µL medium and add 150 µL of fresh Essential 6 medium, containing 100 nM RA and 5% Matrigel
Note
At this moment, you would see some elongation of RA-gastruloids
On day 9 (+120h from RA-gastruloid induction), you should see the fully elongated RA-gastruloid with neural tube and segmented somites