Apr 16, 2025
iPSC-derived Hepatocyte TF Perturbseq
- Helen Streff1,
- Charles Gersbach1
- 1Duke University
- Gersbach Lab
Protocol Citation: Helen Streff, Charles Gersbach 2025. iPSC-derived Hepatocyte TF Perturbseq. protocols.io https://dx.doi.org/10.17504/protocols.io.8epv5n97nv1b/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: April 15, 2025
Last Modified: April 16, 2025
Protocol Integer ID: 126729
Funders Acknowledgements:
NIH
Grant ID: HG012053
Abstract
This protocol describes methods for performing TF Perturbseq in iPSC-derived hepatocytes.
Materials
accutase (Sigma-Aldrich, SCR005)
mTeSR Plus (Stemcell Technologies, 100-0276)
Rock inhibitor (Stemcell Technologies, 72304)
RPMI-1640 (Gibco, 11875093)
B-27 supplement minus insulin (Gibco, A1895601)
1X Penicillin-Streptomycin (Gibco, 15140122)
CHIR99021 (Stemcell Technologies, 72054; Cayman Chemical, 13122)
Recombinant Human/Murine/Rat Activin A (Peprotech, 120-14P)
versene (Gibco, 15040066)
B-27 supplement (Gibco, 17504044)
DMSO (Sigma-Aldrich, D2650)
BMP4 (Peprotech, 120-05-50UG)
bFGF (Peprotech, 100-18B)
HGF (Peprotech, 100-39H)
HCM Hepatocyte Culture Medium BulletKit plus media (Lonza, CC-3198)
recombinant human oncostatin M (Peprotech, 300-10)
100 nM Dexamethasone (Sigma-Aldrich, D4902)
iPSC-derived hepatocytes
iPSC-derived hepatocytes
Day 0: Using accutase (Sigma-Aldrich, SCR005), WTC11 iPSCs were passaged and lentivirally transduced at a high MOI (~3) onto matrigel-coated plates at a density of 10,000 cells/cm2 in mTeSR Plus (Stemcell Technologies, 100-0276) supplemented with 10 uM Rock inhibitor (Stemcell Technologies, 72304)
Day 1: Media was changed with mTeSR Plus (Stemcell Technologies, 100-0276)
Days 2-3: Media was changed with mTeSR Plus (Stemcell Technologies, 100-0276) supplemented with 1 ug/mL puromycin
Day 4: Using accutase (Sigma-Aldrich, SCR005), hPSCs were passaged onto matrigel-coated plates at a density of 20,000 cells/cm2 in mTeSR Plus (Stemcell Technologies, 100-0276) supplemented with 10 uM Rock inhibitor (Stemcell Technologies, 72304)
Days 5-7: Within the first 16 hours, media was replaced and maintained for 3 days with definitive endoderm media consisting of RPMI-1640 (Gibco, 11875093) supplemented with 2% B-27 supplement minus insulin (Gibco, A1895601), 1X Penicillin-Streptomycin (Gibco, 15140122), 3 uM CHIR99021 (Stemcell Technologies, 72054; Cayman Chemical, 13122), and 100 ng/mL Recombinant Human/Murine/Rat Activin A (Peprotech, 120-14P)
Day 8: Wells were briefly (~30 seconds) rinsed with versene (Gibco, 15040066) before adding hepatic specified media consisting of RPMI-1640 (Gibco, 11875093) supplemented with 2% B-27 supplement (Gibco, 17504044), 1X Penicillin-Streptomycin (Gibco, 15140122), 0.5% DMSO (Sigma-Aldrich, D2650), 20 ng/ml BMP4 (Peprotech, 120-05-50UG), and 10 ng/ml bFGF (Peprotech, 100-18B)
Days 9-12: Media was changed with hepatic specified media
Days 13-17: Cells were then maintained for 5 days with daily media changes in hepatoblast media made of RPMI-1640 (Gibco, 11875093) supplemented with 2% B-27 supplement (Gibco, 17504044), 1X Penicillin-Streptomycin (Gibco, 15140122), 0.5% DMSO (Sigma-Aldrich, D2650), and 20 ng/ml HGF (Peprotech, 100-39H)
Days 18-22: Cells were maintained for 5 days with daily media changes in mature hepatocyte media consisting of HCM Hepatocyte Culture Medium BulletKit plus media (Lonza, CC-3198) omitting EGF supplemented with 0.5% DMSO (Sigma-Aldrich, D2650), 20 ng/mL recombinant human HGF (Peprotech, 100-39H), 20 ng/mL recombinant human oncostatin M (Peprotech, 300-10), and 100 nM Dexamethasone (Sigma-Aldrich, D4902)
Prepare cells for scRNAseq
Prepare cells for scRNAseq
Day 23:
o Washed 48 wells 1X with PBS
o Added 1 mL of Tryple-express to each well
o Let sit 25 minutes at 37C
o Resuspended with gentle pipetting into 50 mL tube filled with mature hepatocyte media
o Filtered through 70 um filter
o Spun at 0.3 RCF for 5 minutes
o Resuspended in 3 mL 0.04% BSA (made from 10% stock and PBS) and counted
o Spun again at 0.3 RCF for 5 minutes
o Resuspended with 0.04% BSA and filtered through 30 uM filter