Year: 2023
Type: preprint
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 but do not expand human and mouse HSCs ex vivo. Here we performed a high throughput screen and identified translation inhibitors that allow ex vivo expansion of human HSCs while minimizing cytokine exposure. Transplantation assays show a ∼5-fold expansion of long-term HSCs from UCB after one week of culture in low cytokine conditions. Single cell transcriptomic analysis demonstrates maintenance of HSCs expressing mediators of the unfolded protein stress response, further supporting the importance of regulated proteostasis in HSC maintenance and expansion. These culture conditions maintain adult HSCs after CRISPR/Cas9 editing of the BCL11A+58 enhancer, overcoming a major obstacle to ex vivo gene correction for human hemoglobinopathies. Significance We report ex vivo expansion of human hematopoietic stem cells from umbilical cord blood using low cytokines and small molecule inhibitors of GSK-3, mTORC1, and the translation initiation factor eIF4E. Our method maintains human HSCs ex vivo after gene editing with conventional CRISPR/Cas9, lipid nanoparticle delivery, and base editing approaches, overcoming a major obstacle to gene editing for inherited blood disorders.
Authors: Chenchen Li, Hanna Shin, Dheeraj Bhavanasi, Mai Liu, Xiang Yu, Xiaolei Liu, Friederike Herbst, Mohamad-Gabriel Alameh, Laura Breda, Narasaiah Kovuru, Yeipyeng Kwa, Nithin Sam, Stefano Rivella, Juan R. Alvarez-Dominguez, Gerd A. Blobel, Brian D. Gregory, Scott A. Peslak, Jian Huang, Peter S. Klein
Language: English
Subfield: Hematology
Field: Medicine
Domain: Health Sciences
Open Access status: green
Funders: University of Pennsylvania, Institute for Translational Medicine and Therapeutics, Human Islet Research Network, National Institutes of Health, Perelman School of Medicine, University of Pennsylvania, National Heart, Lung, and Blood Institute, National Institute of Diabetes and Digestive and Kidney Diseases
Awards: Targeting Wnt and mTOR to expand hematopoietic stem cells, G3171598069, Maintenance and expansion of long-term hematopoietic stem cells, G5657272173, Circadian Control of Pancreatic Beta-cell Maturation, Charting human islet maturation via combined soft nanoelectronics and single-cell spatial transcriptomics, Regulation of Hemoglobin Switching, Renewal of the Human Islet Research Enhancement Center (HIREC) for the Type-1-Diabetes-Focused Human Islet Research Network (HIRN)., Expand human umbilical cord blood hematopoietic stem cells with PPAR-a agonists, The role of GSK3/PPAR-/mitophagy pathway in regulating hematopoia, Cellular Signaling Pathways in the Regulation of Fetal Hemoglobin for Treatment of Sickle Cell Disease, Stem Cell and Transplantation Biology
OpenAlex ID: W4389154895