TITLE: Human RA-gastruloid induction from pluripotent stem cells
AUTHORS: Nobuhiko Hamazaki

[DESCRIPTION]
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]
SECTION: Day0, Passage human PSCs onto vitronectin-coated NutriStem
1. Coat wells with Vitronectin and incubate them in 37C for at least 15 min
SECTION: Day0, Passage human PSCs onto vitronectin-coated NutriStem
2. Dissociate pre-treated ESCs
SECTION: Day0, Passage human PSCs onto vitronectin-coated NutriStem
3. Aspirate media from wells and wash wells with PBS(-) and aspirate PBS (-)
SECTION: Day0, Passage human PSCs onto vitronectin-coated NutriStem
4. Add 500 µL Accutase to each well and incubate them at 37C for 4 min
SECTION: Day0, Passage human PSCs onto vitronectin-coated NutriStem
5. 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
SECTION: Day0, Passage human PSCs onto vitronectin-coated NutriStem
6. Transfer them to a new 15mL tube and centrifuge for 3 min at 300 g
SECTION: Day0, Passage human PSCs onto vitronectin-coated NutriStem
7. Suspend them in NutriStem containing 10 µM Y-27632 and spread 2 × 104 cells for 1/12 well onto each vitronectin-coated well
 

SECTION: Day 1-3, Pre-treatment of ESCs (CHIR stimulation)
8. On day 1, Change medium to fresh NutriStem medium containing 5 µM Y-27632
SECTION: Day 1-3, Pre-treatment of ESCs (CHIR stimulation)
9. On day 2, Change the medium to fresh NutriStem medium containing 4 µM CHIR
 

SECTION: Day 1-3, Pre-treatment of ESCs (CHIR stimulation)
10. On day 3, Change medium to fresh NutriStem medium containing 4 µM CHIR + 500 nM RA
 

SECTION: Day 4-9, Human RA-gastruloid induction
11. On day 4, prepare the following reagents

 

SECTION: Day 4-9, Human RA-gastruloid induction
12. Aspirate media from wells and wash wells with PBS(-) and aspirate PBS (-)
SECTION: Day 4-9, Human RA-gastruloid induction
13. Add 500µL Accutase to each well and incubate them in 37C for 4 min
SECTION: Day 4-9, Human RA-gastruloid induction
14. 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
SECTION: Day 4-9, Human RA-gastruloid induction
15. Transfer them to a new 15 mL tube and centrifuge for 3 min at 300 g
SECTION: Day 4-9, Human RA-gastruloid induction
16. Wash again with 2 mL Essential6 + 1µM CHIR + Y and centrifuge it again
SECTION: Day 4-9, Human RA-gastruloid induction
17. Resuspend them in 1-2 mL Essential 6 + Y medium and count cells by Countess
SECTION: Day 4-9, Human RA-gastruloid induction
18. Transfer the necessary number of cells (4000 cells/well) into a reservoir containing a medium (50 µl/well)
SECTION: Day 4-9, Human RA-gastruloid induction
19. Spread cells with a multi-channel pipette
SECTION: Day 4-9, Human RA-gastruloid induction
20. Incubate them in 37C incubator
SECTION: Day 4-9, Human RA-gastruloid induction
21. On day 5 (+24h from RA-gastruloid induction), add 150 µL Essential 6 media to each well.
SECTION: Day 4-9, Human RA-gastruloid induction
22. 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

 

SECTION: Day 4-9, Human RA-gastruloid induction
23. On day 9 (+120h from RA-gastruloid induction), you should see the fully elongated RA-gastruloid with neural tube and segmented somites