Jan 24, 2025

Public workspaceJAX DPC:Differentiation of human induced pluripotent stem cells into extra-embryonic mesenchyme cells

 Forked from JAX DPC: Differentiation of human induced pluripotent stem cells to primitive syncytium
DOI
https://dx.doi.org/10.17504/protocols.io.36wgqdynkvk5/v1
  • Zukai Liu1,
  • Jessica Garofalo1,
  • Juliana Alcoforado Diniz1,
  • Paul Robson1,2
  • 1The Jackson Laboratory for Genomic Medicine;
  • 2UCONN Health
  • MorPhiC Consortium
Juliana Alcoforado Diniz
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DOI: https://dx.doi.org/10.17504/protocols.io.36wgqdynkvk5/v1
Protocol Citation: Zukai Liu, Jessica Garofalo, Juliana Alcoforado Diniz, Paul Robson 2025. JAX DPC:Differentiation of human induced pluripotent stem cells into extra-embryonic mesenchyme cells. protocols.io https://dx.doi.org/10.17504/protocols.io.36wgqdynkvk5/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: January 21, 2025
Last Modified: January 24, 2025
Protocol Integer ID: 118842
Keywords: cell type within the trophoblast lineage, cell lineage, induced pluripotent stem cell, pluripotent stem cell, trophoblast lineage, markers specific to trophoblast, other gene, classic mesenchymal marker, activina to the differentiation media, cell type, cell, cells by the addition, trophoblast, differentiation media
Funders Acknowledgements:
JAX MorPhiC DPC
Grant ID: 1 UM1 HG012651-01
Abstract
This protocol describes how to differentiate hiPSC into extra-embryonic mesenchyme, a cell type within the trophoblast lineage. This 6-day protocol successfully differentiates cells by the addition of BMP4, SU-5402 and ActivinA to the differentiation media. This cell lineage is defined by an essential regulator, HAND1, and expresses classic mesenchymal markers (e.g. COL1A1, COL3A1, LUM) plus markers specific to trophoblast (e.g PTN) and other genes driven by placental- and ape-specific LTR2B.
Troubleshooting
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
ActivinA R&D Catalog #338-AC-050/CF
RevitaCell‱ Supplement (100X) Gibco Catalog #A2644501 
y27632 Stemcell Technologies Catalog #72304
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:
Differentiation of iPSC into fibroblast of villous mesenchyme:
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.
  1. 12. Day 2: Remove the media and add TE media with BMP4 (100ng/ml), SU5402 (20 uM) and ActivinA (100ng/ml).
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 ActivinA (100ng/ml).
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.