Jan 17, 2025
JAX DPC: Differentiation of human induced pluripotent stem cells to primitive syncytium
- Zukai Liu1,
- Jessica Garofalo1,
- Juliana Alcoforado Diniz1,
- Paul Robson1,2
- 1The Jackson Laboratory for Genomic Medicine;
- 2UCONN Health
- MorPhiC Consortium
Protocol Citation: Zukai Liu, Jessica Garofalo, Juliana Alcoforado Diniz, Paul Robson 2025. JAX DPC: Differentiation of human induced pluripotent stem cells to primitive syncytium . protocols.io https://dx.doi.org/10.17504/protocols.io.q26g7mwekgwz/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 routinely use this protocol and it's working
Created: January 16, 2025
Last Modified: January 17, 2025
Protocol Integer ID: 118579
Funders Acknowledgements:
JAX MorPhiC DPC
Grant ID: 1 UM1 HG012651-01
Abstract
This protocol describes how to differentiate hiPSC into primitive syncytium, a cell type within the trophoblast lineage. This 6-day protocol successfully differentiates cells by the addition of BMP4, SU-5402 and A-83 to the differentiation media. The end product has significant syncytialization, though not 100%, and these cells are able to make copious amounts of chorionic gonadotropin (CGA is the most highly expressed gene based on RNA-seq) and have many other gene expression features unique to the syncytial cells of the trophoblast lineage (e.g. PGF, CYP19A1, GCM1, ERVFRD-1, ERVW-1). The resulting syncytial cells are also positive for PRR9 which is unique to the primitive syncytium and absent in the syncytiotrophoblast of the more mature placental villi.
Reagents:
Reagents:
DMEM/F-12 Gibco‱: Catalog #11320033
Knockout Serum Replacement Thermofisher: Catalog #10828028NEAA
L-Glutamine (200 mM) Gibco: Catalog #25030081
MEM Non-Essential Amino Acids Solution (100X) Gibco‱: Catalog #11140050
ß-Mercaptoethanol Gibco: Catalog #21985023
Accutase StemCell Technologies: Catalog #7922
StemFlex Media Fisher: Catalog #A3349401
Trypan Blue Thermo Fisher Scientific: Catalog #15250061
Recombinant Human BMP-4 Protein R&D: Catalog #314-BP-0500/CF
SU-5402 Milipore Sigma: Catalog #572630A-83
BMP4 R&D: Catalog#314-BP-050/CF
RevitaCell‱ Supplement (100X) Gibco: Catalog #A2644501
y27632 Stemcell Technologies: Catalog #72304
A83-01 Tocris: Catalog #2939
Media preparation:
Media preparation:
TE base media for a final volume of 50ml:
DMEM/F12: 39 mL
Knockout Serum: 10 mL
Non-Essential Amino Acids Solution (100x): 500 uL
L-Glutamine (100x): 500 uL
ß-Mercaptoethanol (1000x): 50uL
Procedure:
Procedure:
Differentiation of iPSC into primitive syncytium:
When induced pluripotent stem cells reach confluence in a 6 well plate wash 2x with 3mL of PBS
Remove the PBS and add 1.5mL of accutase and incubate for 7 min at 37°C
Remove the accutase and add complete Stemflex media with Rock inhibitor- Revita Cells (100x) or the Y27632 (10 nM).
Detach the cell from the bottom of the well using a cell lifter.
Transfer the media with the cell suspension to a 15mL tube and centrifuge for 3 min at 300g.
Remove supernatant and add 1ml of media to resuspend the cell pellet.
Count the cell with Typhan Blue in the Cell Countess.
Seed 25k cell/well in a 6-well plate with Stemflex media + Rock inhibitor.
Incubate* at 37°C for 24 hours.
10. Day 0: Remove the Stemflex media and add TE media with BMP4 (100ng/ml) and SU5402 (20 uM).
11. Incubate at 37°C for 48 hours.
12. Day 2: Remove the media and add TE media with BMP4 (100ng/ml), SU5402 (20 uM) and A-83 (1uM).
13. Incubate at 37°C for 48 hours.
14. Day 4: Remove the media and add TE media with BMP4 (100ng/ml), SU5402 (20 uM) and A-83 (1uM).
15. Remove 2ml of media and with a cell lifter harvest the cells from the well.
16. Centrifuge for 3 min at 300g.
17. Remove the supernatant and freeze the pellet for RNA further extraction.
*All incubations are performed with 20% oxygen, unless stated hypoxic condition, in this case, it is 3% oxygen.