Apr 16, 2026

Method for enhancing reprogramming towards induced human expanded pluripotency with engineered SOX17 transcription factors

Communications Biology
DOI
https://dx.doi.org/10.17504/protocols.io.6qpvrynrzgmk/v1
  • Haoqing HU1,2,
  • Derek Hoi Hang Ho1,2,
  • Shi Wing Yeung1,2,
  • Yuebin Tan1,2,
  • Sik Yin Ho2,3,
  • Mingxi Weng2,4,
  • Degong Ruan1,5,
  • Ralf Jauch1,2
  • 1InnoHK Centre for Translational Stem Cell Biology, Hong Kong Science Park, Hong Kong SAR, China;
  • 2School of Biomedical Sciences, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China;
  • 3Laboratory for Primate Embryogenesis, Department of Physiology, Development and Neuroscience, University of Cambridge, Downing Street, Cambridge, UK;
  • 4Altos Labs, San Diego, CA 92122, USA;
  • 5The University of Hong Kong-Shenzhen Hospital Translational Medicine Research Centre, Shenzhen, China
  • Haoqing Hu -- Ralf Jauch lab
Haoqing HU
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DOI: https://dx.doi.org/10.17504/protocols.io.6qpvrynrzgmk/v1
External link: https://doi.org/10.1038/s42003-026-09963-7
Protocol Citation: Haoqing HU, Derek Hoi Hang Ho, Shi Wing Yeung, Yuebin Tan, Sik Yin Ho, Mingxi Weng, Degong Ruan, Ralf Jauch 2026. Method for enhancing reprogramming towards induced human expanded pluripotency with engineered SOX17 transcription factors. protocols.io https://dx.doi.org/10.17504/protocols.io.6qpvrynrzgmk/v1
Manuscript citation:
Haoqing Hu, Derek Hoi Hang Ho, Shi Wing Yeung, Yuebin Tan, Sik Yin Ho, Mingxi Weng, Degong Ruan, Ralf Jauch (2026) Enhancing reprogramming towards induced human expanded pluripotency through substitution of SOX2 with engineered SOX17 transcription factors.Communications Biology doi: 10.1038/s42003-026-09963-7
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: March 04, 2026
Last Modified: April 16, 2026
Protocol  Integer ID: 246258
Keywords: reprogramming, stem cell, transcription factors, engineered sox17 transcription factor, sox17 transcription factors this protocol, sox17 transcription factor, expanded potential stem cell, potential stem cell, expanded pluripotency
Funders Acknowledgements:
InnoHK initiative of the Innovation and Technology Commission of the Hong Kong Special Administrative Region Government
Grant ID: InnoHK initiative of the Innovation and Technology Commission of the Hong Kong Special Administrative Region Government
Research Grants Council of Hong Kong General Research Fund
Grant ID: 17106622
Research Grants Council of Hong Kong General Research Fund
Grant ID: 17117925
Research Grants Council of Hong Kong General Research Fund
Grant ID: 17101120
Applied Stem Cell Conservation Fund
Grant ID: Applied Stem Cell Conservation Fund
Disclaimer
The authors declare the following competing interests: H.H., D.H.H.H., M.W., and R.J. are inventors on U.S. Patent Application No. 18/453,112 (published as US 2024/0084265 A1) relating to this work. The authors may be entitled to royalty or other financial benefits under their institutions’ IP policies. All other authors declare no competing interests.
Abstract
This protocol describes a highly efficient method for reprogramming human induced expanded potential stem cells (iEPSCs) using engineered SOX17 transcription factors.
Materials
Cell Source

  • Human HDF adult (Thermo Fisher Scientific, Cat #: C-013-5C)
  • Human HDF neonatal cells (Thermo Fisher Scientific, Cat #: C-004-5C)
  • SNL76/7 cells (ATCC, Cat #: SCRC-1049)
  • HEK293T cells

Plasmids

  • M2rtTA (Addgene, #20342)
  • psPAX2 (Addgene, #12260)
  • pMD2.G (Addgene, #12259)
  • FUW-tetO-hOKMS (Addgene, #51543)
  • FUW-tetO-hOKMSOX17FNV (Addgene, #206382) [1]
  • FUW-tetO-hOKMminiSOX (Addgene, #206383) [1]

Reagents and Kits

  • Mitomycin-C (Stemcell, #73274)
  • TrypLETM Express Enzyme (Life Technologies, #12605028)
  • Dulbecco’s Modified Eagle Medium (DMEM, Gibco, #12100046)
  • Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F-12, Gibco, #21331020)
  • Neurobasal medium (Gibco, #21103049)
  • Phosphate-buffered saline (PBS, Gibco, #10010023)
  • Dimethyl sulfoxide (DMSO, Sigma, #D2650-100ML)
  • Fetal bovine serum (FBS, Gibco, #10270106)
  • Knockout DMEM (Gibco, #10829018)
  • 100X GlutaMAX (Gibco, #35050061)
  • 100X non-essential amino acids (NEAA, Gibco, #11140050)
  • Penicillin–streptomycin (Gibco, #15140122)
  • Minimum Essential Medium (MEM, Thermo Fisher Scientific, #11-095-080)
  • 2-mercaptoethanol (Life Technologies, #21985023)
  • Geltrex (Life Technologies, #A1413302)
  • Matrigel (Corning, #354277)
  • KnockOut Serum Replacement (KSR, Gibco, #10828028)
  • Activin A (MCE, # HY-P70311)
  • Y-27632 (MCE, # HY-10071)
  • 100X Penicillin–streptomycin-glutamine (Gibco, #10378016)
  • 100X N2 (Gibco, #17502001)
  • 50X B27 (Gibco, #17504001)
  • 100X Sodium pyruvate (Gibco, #11360070)
  • 100X ITS-X (Gibco, #5150056)
  • 7.5% Bovine albumin fraction V (Gibco, #15260037)
  • L-Ascorbic acid 2-phosphate sesquimagnesium salt hydrate (LAAP, Sigma, #A8960)
  • 500X Lipid concentrate (Gibco, #11905031)
  • 1000X Trace elements B (Corning, #25022CI)
  • 1000X Trace elements C (Corning, #25023CI)
  • Glutathione reduced (Sigma, #G4251-300MG)
  • XAV-939 (MCE, #HY-15147)
  • IWR-1-endo (MCE, #HY-12238)
  • CHIR-99021 (MCE, #HY-10182)
  • A-419259 (MCE, #HY-15764)
  • hLif (MCE, # HY-P73276)
  • Human EGF (Gibco, # PHG0313)
  • A83-01 (Tocris, #2939/10)
  • SB431542 (MCE, #HY-10431)
  • Valproic Acid (VPA, Stemcell, #72292)
  • Linear polyethyleneimine (PEI, Polysciences, #23966)
  • Polybrene (Sigma, #40804ES76)
  • Doxycycline (DOX, Sigma-Aldrich, #D9891-1G)
  • Paraformaldehyde (PFA, Beyotime, # P0099-500ml)
  • Triton X-100 (Sigma, #X100-500ML)
  • bovine serum albumin (BSA, USBIOLOGICAL, #A1311-100G)
  • NucBlue Fixed Cell Stain (Life Technologies, #R37606)
  • TRIzol (Invitrogen, #15596018)
  • Sodium Butyrate (NaB, Sigma, #B5887-1G)

Suggested antibodies
ABCD
Antibodies Source Identifier Working concentration
Mouse anti-CD75 Biolegend 326901 1:100
Goat anti-OCT4 Santa Cruz Sc-8628 1:500
Rabbit anti-NANOG Novus Biologicals NB100-58842 1:100
Mouse anti-SSEA4 Santa Cruz Sc-21704 1:500
Mouse anti TRA-1-60 Sigma-Aldrich MAB4360 1:500
Rabbit anti-FOXA2 Cell Signaling 8186T 1:500
Mouse anti-αSMA Sigma-Aldrich A2547 1:400
Mouse anti TUJ1 Biolegend 801212 10ug/mL
Mouse anti GATA3 R&D Systerms MAB6330 10ug/mL
Goat anti GATA2 R&D Systerms AF2046 5ug/mL
Rabbit anti-KRT7 Abcam ab181598 1:100
Donkey anti Mouse Alexa Fluor 488 Thermo Fisher Scientific A-21202 1:1000
Donkey anti Goat Alexa Fluor 488 Thermo Fisher Scientific A-11055 1:1000
Donkey anti Rabbit Alexa Fluor 488 Thermo Fisher Scientific A-21206 1:1000
Donkey anti Rabbit Alexa Fluor 594 Thermo Fisher Scientific A32754 1:1000
Donkey anti Mouse Alexa Fluor 594 Thermo Fisher Scientific A-21203 1:1000
Medium Setup

  • 293T medium: DMEM supplemented with 10% FBS. Store at 4 °C for up to 1 month. Equilibrate to room temperature before use.
  • M10 medium: Knockout DMEM, 10% FBS, 1% GlutaMAX, 1% NEAA, 1% penicillin–streptomycin. Store at 4 °C for up to 1 month. Equilibrate to room temperature before use.
  • Human dermal fibroblast medium (HDF medium): MEM, 15% FBS, 1% GlutaMAX, 1% NEAA, 1% penicillin–streptomycin, 100 µM 2-mercaptoethanol. Store at 4 °C for up to 1 month. Equilibrate to room temperature before use.
  • Human expanded potential stem cell medium (hEPSCM), modified from [2]: DMEM/F-12 and Neurobasal medium are mixed at a 1:1 ratio, supplemented with 1x penicillin–streptomycin-glutamine, 1x MEM NEAA, 0.5x N2, 1x B27, 1x Sodium pyruvate, 1x ITS-X, 0.045% bovine albumin fraction V, 1x β-mercaptoethanol, 64 µg per mL LAAP, 1x chemically defined lipid concentrate, 1x trace elements B, 1x trace elements C, 3.3 µg per mL l-glutathione reduced, 1 µM XAV-939, 2.5 µM IWR-1-endo, 1 µM CHIR-99021, 0.1 µM A-419259. Store at 4 °C for up to 1 month. Equilibrate to room temperature before use.
  • Feeder-free hEPSCM: hEPSCM supplemented with 3% KnockOut Serum Replacement, 20 ng per mL Activin A, 2.5 µM Y-27632. Y-27632 should be added fresh immediately before use. Store at 4 °C for up to 1 month. Equilibrate to room temperature before use.
  • Trophoblast stem cell medium (TSCM) [3]: DMEM/F12, 0.2% FBS, 1% penicillin–streptomycin, 100 µM 2-mercaptoethanol, 0.3% BSA, 1x ITS-X, 1.5  μg per mL LAAP, 50 ng per mL EGF, 2 μM CHIR99021, 0.5 μM A83-01, 1 μM SB431542, 0.8 mM VPA.
  • M15 medium: Knockout DMEM, 15% FBS, 1% GlutaMAX, 1% NEAA, 1% penicillin–streptomycin, 100 μM 2-mercaptoethanol.
  • VLD medium: M15 medium supplemented with 50 μg per mL LAAP, 10 ng per mL hLif and DOX.
  • DFK medium: DMEM/F-12 supplemented with 20% Knockout Serum Replacement, 10 μM Y-27632.

Plate preparation

  • Coat tissue-culture plates with 0.2% gelatin in sterile water for at least 30 min at room temperature (or 37 °C). Aspirate gelatin immediately before use (no need to rinse).
  • For feeder-free conditions, thaw Geltrex/Matrigel on ice at 4 °C, dilute in cold DMEM/F-12 to the working concentration (e.g., 1:100; optimize per lot), coat plates (e.g., 1 h at 37 °C), and keep coated plates at 37 °C until seeding. Aspirate matrix containing medium immediately before use (no need to rinse).
  • For feeder plate preparation, thaw mitomycin-C–inactivated SNL76/7 feeders rapidly at 37 °C, dilute in pre-warmed M10 medium, centrifuge at 300 ×g for 3 min, resuspend, and plate on 0.2% gelatin-coated plates. Allow feeders to attach for at least 24 hours before seeding target cells (48 hours are preferred).


Safety warnings
Safety and regulatory compliance
This protocol involves production and use of lentiviral vectors. All procedures with lentivirus (transfection, collection, filtration, storage, transduction, and waste handling) must be performed in a certified Class II biosafety cabinet under BSL-2 (or higher, as required by local regulations). Wear appropriate PPE (lab coat, gloves, eye protection). Decontaminate surfaces and liquid waste with freshly prepared 10% bleach (final) for at least 30 min (or an equivalent institutional disinfectant), followed by disposal according to institutional biosafety guidelines. Sharps should be avoided whenever possible. Users must obtain prior approval from the institutional biosafety committee and follow local regulations for work with human cells and viral vectors.
Cell culture conditions
HEK293T cells were cultured in 293T medium.
Feeder cells (SNL76/7 feeders) were cultured in M10 medium on 0.2% gelatin-coated plates.
The human adult and neonatal dermal fibroblasts (HDFs) were cultured in HDF medium on 0.2% gelatin-coated plates.
Induced expanded potential stem cell (iEPSC) clonal cell lines were maintained on feeder cells in hEPSCM or on Geltrex or Matrigel in feeder-free hEPSCM.
All cells were cultured under normoxic conditions at 37 °C and 5% CO2.

Feeder cell preparation
Active SNL76/7 cells are maintained in M10 medium on 0.2% gelatin-coated plates until they reach 90-100% confluence.
Cells are washed once with PBS.
Incubate cells with M10 medium containing 10 µg per mL mitomycin-C for 4 hours.
Note
Mitomycin-C degrades over time at room temperature and light-sensitive. It is recommended to be used immediately after dissolve.

Wash cells with PBS.
Dissociate into single cells with TrypLE for 3 min at 37 °C and neutralize with M10 medium.
Centrifuge at 300 xg for 3 min, discard the supernatant.
Cryopreserve cell pellets in freezing medium consisting of 90% FBS and 10% DMSO and store in -80 °C for future use.
Lentivirus preparation
HEK293T cells were seeded at 2.5× 106 cells per 60 mm dish and cultured overnight.
On the day of transfection, mix 4 µg of plasmids containing polycistronic reprogramming cassettes or M2rtTA, packaging plasmids: 2.4 µg psPAX2 and 1.6 µg pMD2.G and 16 µg PEI in DMEM. Incubate for 15 min at room temperature, then add the mixture to the cells.
Culture medium was changed to fresh 293T medium containing 1 mM Sodium Butyrate (NaB) 10–14 hours post-transfection.
Lentivirus-containing supernatant was collected 48-, 72- and 96-hours post-transfection, kept at 4 °C and pooled together. Fresh 293T medium without NaB was added back after virus collection.
Cell debris was removed by passing through 0.45 µm filters once and centrifuging at 2000 xg for 10 min at room temperature.
Lentivirus can be aliquoted and stored in -80 °C for up to 1 year.
Human iEPSC reprogramming
Day -2
Dissociate human fibroblasts with TrypLE at 37 °C for 3 min and neutralize with HDF medium.
Centrifuge at 300 xg for 3 min to harvest cell pellets and seed 2-2.5 x 104 cells per 0.2%-gelatin coated well of a 12-well plate in HDF medium.
Day -1
On the day of transduction, for 1 well of a 12-well plate, mix lentivirus-containing supernatant (reprogramming cassette:M2rtTA = 2:1) with polybrene at a final concentration of 8 μg per mL. Bring total volume to 1.0 mL with M15 medium.
Note
Virus volume (guideline): Depending on viral titer, add 150–300 µL of unconcentrated reprogramming-cassette lentiviral supernatant per well (12-well format), plus M2rtTA lentivirus at half that volume (i.e., a 2:1 reprogramming cassette:M2rtTA volume ratio). If transduction efficiency is low, increase viral volume; if cytotoxicity is high, reduce viral volume.

Note
Only one type of polycistronic reprogramming cassette is required. Choose either the engineered SOX cassette for highly efficient reprogramming, or the standard SOX2 cassette for routine reprogramming.

Aspirate HDF medium completely and immediately add 1.0 mL transduction mix to each well. Incubate overnight.
Day 0
Medium was changed to fresh VLD medium (0.5 µg per mL DOX for recovery).
Note
If no significant cell death is observed over the next 2–6 days, the recovery step can be omitted and DOX can be used at 1 µg per mL directly.

Day 1
Medium was changed to fresh VLD medium (1 µg per mL DOX).
Day 2 to Day 6
Change medium with fresh VLD medium (1 µg per mL DOX) every day.
Note
Transduced fibroblasts should remain adherent with limited cell death. Excessive detachment suggests viral toxicity or polybrene sensitivity.

Day 7
Dissociate the transduced fibroblasts with TrypLE at 37 °C for 3 min and neutralize with M10 medium.
Centrifuge and re-plate cell pellets onto feeder cells (feeder-based) or Geltrex (feeder-free) at the density of 1.5 x 104 live cells per well of a 12-well plate in VLD medium (1 µg per mL DOX).
Day 8 to Day 15
Change medium with fresh VLD medium (1 µg per mL DOX) every day.
Note
Compact, dome-shaped cell clusters should begin to emerge (timing may vary by donor and passage). If no or very few colonies emerge, verify virus titer, transduction efficiency, medium composition, DOX activity, feeder quality, and matrix coating.

Day 16
Change medium to hEPSCM supplemented with 1 µg per mL DOX.
Change medium every day to hEPSCM supplemented with 1 µg per mL DOX for another 3-5 days until the dome-shaped iEPSC colonies reach a desired size and shape.
Culture the cells with DOX-free hEPSCM for another 2 days before colony counting. The total culture duration in hEPSCM should be at least 5 days.
For colony counting, fix cultures with 4% PFA on the last day. Perform immunocytochemistry (ICC) and stain with an anti-CD75 antibody. CD75-positive colonies with dome-shaped morphology were counted.
Colony picking and establishment of clonal iEPSC cell lines
Manually pick the dome-shaped colonies at the end stage of reprogramming. For feeder-based conditions, follow sub-steps 1–5; for feeder-free conditions, follow sub-steps 6–8.
Picked-out feeder-based colonies were pipetted into small pieces and seeded onto fresh feeder layers in hEPSCM with 10 µM Y-27632 for the first 24 hours.
Change medium every day with hEPSCM without Y-27632 until the colonies reach a desired size.
Passage the above cultures as single cells onto new feeder layers.
When the sub-clones reach a desired size, repeat the colony picking procedures for two additional rounds.
After the third round of colony picking, passage cultures with TrypLE to expand for validation or cryopreservation.
Picked-out feeder-free colonies were pipetted into small pieces and seeded onto Geltrex or Matrigel-coated plates in feeder-free hEPSCM with 10 µM Y-27632 for the first 24 hours.
Change medium every day with feeder-free hEPSCM with 2.5 μM Y-27632 until the colonies reach a desired size.
Because feeder-free iEPSCs thrive in low-density single-cell conditions, colony picking was performed only once. The isolated colonies were subsequently transferred onto Geltrex- or Matrigel-coated plates and expanded for downstream validation or cryopreservation.
Immunocytochemistry (ICC)
Cells were fixed with 4% PFA for 15 minutes at room temperature, permeabilized in PBS with 0.2% Triton X-100 for 10 minutes at room temperature and blocked with 5% BSA in PBS for 2 hours at room temperature.
The primary antibody diluted in 1% BSA in PBS was incubated overnight at 4 °C. (NANOG, OCT4, SSEA4, TRA-1-60)
The samples were then washed three times for 5 minutes with PBS. Secondary antibodies diluted in 1% BSA in PBS were added and incubated for 1 hour at room temperature. Nuclei were counterstained with NucBlue Fixed Cell Stain.
In vitro tri-lineage embryonic differentiation of human iEPSCs
Human iEPSCs were dissociated into single-cell suspensions using TrypLE and seeded at the density of 5000 cells per well of an ultra-low 96-well plate in DFK medium for embryoid bodies (EBs).
If the cells were cultured with feeder layers, feeders were removed by incubating in 0.2% gelatin-coated wells at 37 °C for at least 45 min. Loosely attached cells were harvested and used for EB formation.
On day 2, add 100 µL of fresh DFK medium per well.
On day 4, around 6 EBs per well were transferred to a Geltrex-coated 24-well plate in DFK medium.
Following an additional 12-14 days with medium change every other day, cultures were fixed with 4% PFA and stained with lineage markers TUJ1, α-SMA, and FOXA2.
Evaluation of extra-embryonic differentiation capacity of iEPSCs
To derive trophoblast stem cells (TSCs) from the iEPSC clones, cells were dissociated into a single-cell suspension with TrypLE and seeded on the Matrigel or Geltrex-coated plates in TSCM.
If the cells were cultured with feeder layers, feeders were removed by incubating in 0.2% gelatin-coated wells at 37 °C for at least 45 min. Loosely attached cells were harvested and used for seeding.
Change medium with fresh TSCM every other day for around 1 week until TSC-like morphology emerged.
Fix the cultures with 4% PFA and stain for KRT7, GATA2, GATA3, and NANOG.
Protocol references
[1] Hu, H. et al. Evaluation of the determinants for improved pluripotency induction and maintenance by engineered SOX17. Nucleic Acids Res 51, 8934-8956, doi:10.1093/nar/gkad597 (2023).

[2] Ruan, D. et al. Establishment of human expanded potential stem cell lines via preimplantation embryo cultivation and somatic cell reprogramming. Nature Protocols 20, 2698-2734, doi:10.1038/s41596-025-01168-2 (2025).

[3] Okae, H. et al. Derivation of Human Trophoblast Stem Cells. Cell Stem Cell 22, 50-63.e56, doi:10.1016/j.stem.2017.11.004 (2018).