Date of Award
Doctor of Philosophy (PhD)
Cell & Molecular Biology
The transfusion of healthy hematopoietic cells is required to treat diseases of and injury to the hematopoietic system. Hematopoietic stem cell transplantation (HSCT) is the only curative treatment for a variety of both malignant and non-malignant hematological disorders arising from dysfunction of hematopoietic stem cells (HSCs). HSCT is limited by our inability to robustly expand human HSCs ex vivo, especially for autologous gene therapy-based HSCT for monogenic disorders. Therefore, developing strategies that expand HSCs could greatly improve HSCT, particularly for gene therapy approaches for bone marrow failure (BMF) syndromes with defective HSCs such as Fanconi Anemia (FA). This thesis investigates the role of the adaptor protein LNK (SH2B3) in human hematopoiesis and whether depletion of LNK in human hematopoietic stem and progenitor cells (HSPCs) would expand human HSCs to improve HSCT. LNK is an established negative regulator of cytokine signaling in murine hematopoietic HSPCs and its deficiency potentiates activation of JAK2. As a result, mice genetically deficient for Lnk have an expanded pool of HSCs with enhanced self-renewal. Here, we demonstrate that depletion of LNK via lentiviral expression of miR30-based short hairpin RNAs (shRNAs) resulted in robust expansion of transplantable human HSCs that provided balanced multilineage reconstitution xenotransplant without evidence of malignant transformation. Importantly, LNK depletion enhanced cytokine mediated JAK/STAT activation in CD34+ HSPCs. Moreover, we demonstrate that LNK depletion expands primary HSPCs associated with FA using FA-like stem cell models and primary patient derived FA HSCs. In addition, we show that genetic deletion of LNK in human pluripotent stem cells increases their production of hematopoietic cells in growth factor driven differentiation protocols. Together, these results demonstrate the potential of targeting LNK to expand HSCs to improve HSCT, HSCT-based gene therapy and potentially other hematopoietic cell therapies.
Holdreith, Nicholas Bryden, "Targeting Lnk (sh2b3) To Expand Healthy And Fanconi Anemia Human Hematopoietic Stem And Progenitor Cells" (2021). Publicly Accessible Penn Dissertations. 5062.