Hematopoietic stem cells (HSCs) are a cornerstone of regenerative medicine, both as a model system to study stem cell biology and therapeutically for HSC transplants (HSCT) in the treatment of bone marrow failure, myeloid neoplasms, and other hematopoietic disorders. In the near future, HSCT will also be a critical step in the application of genome editing to treat inherited blood disorders such as sickle cell disease and thalassemia. However, HSCs are rare cells that rapidly lose their capacity for self-renewal outside of the hematopoietic niche, presenting a major challenge to ex vivo study of HSCs and to the expansion of HSCs for therapeutic applications. HSCT with umbilical cord blood (UCB) donors is widely used in children because of the reduced stringency required for HLA matching and the lower risk of graft versus host disease, but UCB transplants are limited in adults because of the low number of HSCs. Similarly, a major obstacle to genome editing for inherited blood diseases is the low number of HSCs after genome editing and the loss of self- renewing cells after ex vivo manipulation. Therefore, a method to increase HSCs would substantially change the therapeutic landscape in HSCT. We previously established a culture system that maintains long-term HSCs ex vivo. We then performed a high throughput screen of >2200 bioactive compounds for additional small molecules that allow expansion of HSCs and identified a lead compound that confers ~10-fold expansion of HSCs within 4 days of culture, as confirmed by limiting dilution analysis and long-term engraftment in mouse xenografts. This compound also confers expansion of CRISPR modified phenotypic HSCs from adult donors. We have identified additional compounds from the screen that confer ex vivo expansion of phenotypic HSCs but have not yet validated these by xenotransplantation assays.
The specific aims of this proposal are to: 1) rigorously test the in vivo function of HSCs expanded from UCB and CRISPR-modified HSCs; 2) establish the mechanism of our lead compound, an inhibitor of the translation initiation factor eIF4E, in HSC homoestasis; and 3) explore additional compounds identified in the screen, including the development of a novel approach to accelerate the in vivo testing of multiple compounds in mouse xenografts assays. The ability to expand functional HSCs ex vivo will provide a critical therapeutic advance for HSC transplant when the number of HSCs is limiting, including umbilical cord blood and genome-edited HSCs.

Public Health Relevance

Hematopoietic stem cell transplantation is a cornerstone of treatment for bone marrow failure, severe myeloproliferative and myelodysplastic disorders, leukemia, and lymphoma and could also be a new approach to treating inherited blood disorders such as sickle cell disease. This proposal describes a method to increase HSCs in the laboratory so that we can use them for transplantation and to study what controls HSC growth and differentiation.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Project (R01)
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Development - 2 Study Section (DEV2)
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Bai, C Brian
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University of Pennsylvania
Internal Medicine/Medicine
Schools of Medicine
United States
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