Abstract
Hematopoietic stem cell (HSC) transplantation using umbilical cord blood (UCB) is a potentially life-saving treatment for leukemia and bone marrow failure but is limited by the low number of HSCs in UCB. The loss of HSCs after ex vivo manipulation is also a major obstacle to gene editing for inherited blood disorders. HSCs require a low rate of translation to maintain their capacity for self-renewal, but hematopoietic cytokines used to expand HSCs stimulate protein synthesis and impair long-term self-renewal. We previously described cytokine-free conditions that maintain human and mouse HSCs ex vivo using inhibitors of GSK-3 (CHIR) and mTOR (Rapamycin), referred to hereafter as “CR”.
Here we performed a high throughput screen of 2,240 FDA approved and/or bioactive compounds and found that the translation inhibitor 4E1RCat allows ex vivo expansion of human HSCs from UCB in CR medium with minimal cytokine exposure. Single cell transcriptomic analysis demonstrates maintenance of HSCs expressing mediators of the unfolded protein stress response, further supporting the potential importance of regulated proteostasis in HSC maintenance and expansion. We confirmed that these culture conditions promote the expansion of long-term HSCs by limiting dilution analysis (LDA) and secondary transplantation. The LDA shows a ~5-fold expansion of functional HSCs from UCB after one week of culture in CR with low cytokines and 4E1RCat. Secondary transplantation assays confirm expansion of long-term HSCs with multilineage potential.
CRISPR/Cas9 editing of the BCL11A+58 enhancer is now an FDA approved therapy for sickle cell disease and transfusion-dependent β-thalassemia. However, loss of functional HSCs after ex vivo manipulation and culture remains a major obstacle to therapeutic gene editing. Our culture conditions maintain and expand human adult HSCs after gene editing by both electroporation and lipid nanoparticles (LNPs) delivery methods. After 7 days of culture in CR with low cytokines and 4E1RCat, phenotypic HSCs expanded ~35-78-fold compared to day 0 for HSCs undergoing electroporation or exposure to LNPs. Fetal hemoglobin (HbF) expression increased substantially after BCL11A editing in erythroid cells derived from day 7 expansion after electroporation or LNP delivery. Transplant of BCL11A-edited HSCs cultured in CR and low cytokines with 4E1RCat showed high engraftment comparable to freshly isolated HSCs, indicating maintenance of HSCs after culture in these conditions. Culturing edited HSCs under these conditions may therefore overcome a major obstacle to ex vivo gene correction for human hemoglobinopathies by maintaining functional HSCs.
