Human RA-gastruloid induction from pluripotent stem cells
Nobuhiko Hamazaki
Disclaimer
DISCLAIMER – FOR INFORMATIONAL PURPOSES ONLY; USE AT YOUR OWN RISK
The protocol content here is for informational purposes only and does not constitute legal, medical, clinical, or safety advice, or otherwise; content added to protocols.io is not peer reviewed and may not have undergone a formal approval of any kind. Information presented in this protocol should not substitute for independent professional judgment, advice, diagnosis, or treatment. Any action you take or refrain from taking using or relying upon the information presented here is strictly at your own risk. You agree that neither the Company nor any of the authors, contributors, administrators, or anyone else associated with protocols.io, can be held responsible for your use of the information contained in or linked to this protocol or any of our Sites/Apps and Services.
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.
Steps
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
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
On day 3 , Change medium to fresh NutriStem medium containing 4 µM CHIR + 500 nM RA
Day 4-9, Human RA-gastruloid induction
On day 4 , prepare the following reagents
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
On day 9 (+120h from RA-gastruloid induction) , you should see the fully elongated RA-gastruloid with neural tube and segmented somites
