Nov 21, 2025
Protocol for enhancing ex vivo expansion of human haematopoietic stem cells by inhibiting ferroptosis
- Lucrezia della Volpe1,2,3,
- Vijay G. Sankaran1,2,3,4
- 1Division of Hematology/Oncology, Boston Children’s Hospital and Department of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USA;
- 2Howard Hughes Medical Institute, Boston, MA, USA;
- 3Broad Institute of MIT and Harvard, Cambridge, MA, USA;
- 4Harvard Stem Cell Institute, Cambridge, MA, USA.
External link: https://doi.org/10.1038/s41556-025-01814-7
Protocol Citation: Lucrezia della Volpe, Vijay G. Sankaran 2025. Protocol for enhancing ex vivo expansion of human haematopoietic stem cells by inhibiting ferroptosis. protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlkyr8wg5r/v1
Manuscript citation:
della Volpe, L., Lee, A.J., Antoszewski, M. et al. Inhibiting ferroptosis enhances ex vivo expansion of human haematopoietic stem cells. Nat Cell Biol (2025). https://doi.org/10.1038/s41556-025-01814-7
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: October 16, 2025
Last Modified: November 21, 2025
Protocol Integer ID: 229997
Keywords: HSCs, Ferroptosis, ex vivo expansion, inhibiting ferroptosis efficient ex vivo expansion, ex vivo expansion of human haematopoietic stem cell, inhibiting ferroptosi, applying ferroptosis inhibition, ferroptosis inhibition, treatment with ferroptosis inhibitor, ferroptosis inhibitor, human haematopoietic stem cell, human hematopoietic stem cell, ex vivo human hsc expansion, major challenge for transplantation, iron, human hsc expansion, multilineage engraftment in xenotransplantation assay, vivo expansion, xenotransplantation assay, expansion of both cord blood, regulated cell death, transplantation, molecular stem cell feature
Abstract
Efficient ex vivo expansion of human hematopoietic stem cells (HSCs) remains a major challenge for transplantation and genome-engineering applications, as standard culture systems lead to substantial HSC loss. This protocol describes a method to enhance HSC expansion by inhibiting ferroptosis, an iron-dependent form of regulated cell death. Treatment with ferroptosis inhibitors such as liproxstatin-1 (Lip-1) or ferrostatin-1 (Fer-1) significantly improves expansion of both cord blood (CB) and mobilized peripheral blood (mPB)–derived HSCs. Importantly, the expanded cells preserve phenotypic and molecular stem cell features and sustain long-term, multilineage engraftment in xenotransplantation assays. This step-by-step protocol outlines the optimized culture conditions and workflow for applying ferroptosis inhibition to improve ex vivo human HSC expansion and functional maintenance.
For complete details on the results and validation of this protocol, please refer to our work published in Nature Cell Biology.
Troubleshooting
Step-by-step protocol - SFEM II culture for human CD34⁺ HSPCs (with ferroptosis inhibition)
Prepare reagents
Warm the following media to room temperature (RT) or in a 37 °C water bath:
Thawing Medium: PBS + 1% FBS.
SFEM Culture Medium: StemSpan SFEM II (StemCell Technologies) supplemented with:
- 1% L-glutamine (Thermo Fisher Scientific)
- 1% penicillin/streptomycin (Life Technologies)
- 1×CC100 cytokine cocktail (FLT3L, SCF, IL3, IL6; StemCell Technologies)
- 100 ng/mL recombinant thrombopoietin (TPO; PeproTech)
- 35 nM UM171 (StemCell Technologies)
Ferroptosis inhibitors:
- Liproxstatin-1 (Lip-1; Cayman Chemical), prepare a 10 mM stock in DMSO; use at 10 µM final concentration.
- Ferrostatin-1 (Fer-1; MedChemExpress), prepare a 10 mM stock in DMSO; use at 5 µM final concentration.
Thaw CB- or mPB-derived CD34+
Thaw the cryovial in a 37 °C water bath until only a small ice crystal remains. Transfer the cell suspension dropwise into a 15 mL conical tube containing 10 mL of Thawing Medium and centrifuge at 300 × g for 10 min at RT.
Cell culture
Carefully aspirate, discard the supernatant, and resuspend the cell pellet in SFEM Culture Medium supplemented with either 10 µM Lip-1, or 5 µM Fer-1. Count viable cells and adjust the concentration to 5×10⁵ cells/mL and plate cells in an appropriate culture vessel and incubate under standard culture conditions (37 °C, 5% CO₂).
Culture maintenance
Split the cultures every 2–3 days by diluting cells to a final concentration of 3-5×10⁵ cells/mL in fresh SFEM Culture Medium, and continue expansion until the desired end point.
Replenish ferroptosis inhibitors by adding Lip-1 (10 µM) or Fer-1 (5 µM) at each medium change.
Step-by-step protocol - Cytokine-free expansion medium (CFEM) for human CD34⁺ HSPCs (with ferroptosis inhibition)
Prepare reagents
Prepare the medium and warm it to room temperature (RT) or in a 37 °C water bath:
CFEM Culture Medium:
- (Iscove’s Modified Dulbecco’s Medium-based (IMDM) (Life Technologies)
- 1% Insulin-Transferrin-Selenium-Ethanolamine (ITS-X; Life Technologies)
- 1% L-glutamine (Thermo Fisher Scientific)
- 1% Penicillin/Streptomycin (Life Technologies)
- 1 mg/mL Polyvinyl alcohol (PVA; Sigma-Aldrich)
- 1 µM 740Y-P (MedChemExpress)
- 0.1 µM Butyzamide (MedChemExpress)
- 70 nM UM171 (StemCell Technologies)
Ferroptosis inhibitors:
- Liproxstatin-1 (Lip-1; Cayman Chemical), prepare a 10 mM stock in DMSO; use at 10 µM final concentration.
- Ferrostatin-1 (Fer-1; MedChemExpress), prepare a 10 mM stock in DMSO; use at 5 µM final concentration.
Cell isolation
Isolate CD34⁺ HSPCs from cord blood using the EasySep Human Cord Blood CD34⁺ Positive Selection Kit (StemCell Technologies) following the manufacturer’s instructions.
Cell culture
Count viable freshly isolated CD34+ cells using trypan blue or an equivalent viability dye and adjust the final cell concentration to 7×10⁴–1×10⁵ cells/mL in CFEM Culture Medium supplemented with either 10 µM Lip-1 or 5 µM Fer-1. Seed CD34⁺ HSPCs into CellBind plates, using 1 mL of medium per well for 24-well (Fisher Scientific, 08757216) plates or 5 mL of medium per well for 6-well plates (Corning, 3335).
Culture maintenance
Split cultures every 3–4 days to maintain a density of 7×10⁴–1×10⁵ cells/mL. Gently mix the culture to fully resuspend the cells, count them, and transfer an appropriate volume into fresh CFEM medium containing Lip-1 or Fer-1, replenishing the ferroptosis inhibitors with each medium change to maintain constant exposure.