Regulation of human hematopoietic stem cell lineage commitment
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Understanding the molecular mechanisms that govern human hematopoiesis remains a major goal of both developmental and clinical biology. My thesis projects focus on identifying and characterizing regulatory factors involved in myeloid commitment steps for human hematopoietic stem and progenitor cells (HSPCs), so as to expand them in vitro or direct them into desired lineages. I first studied the histone methyltransferases G9a and GLP, which play key roles during mammalian development through mono- and di-methylation of histone H3 lysine 9 (H3K9me1/2), modifications associated with transcriptional repression. I found that G9a/GLP activity drives progressive, genome-wide H3K9me2 patterning in euchromatin during HSPC lineage specification. Remarkably, HSPCs continuously treated with UNC0638, a G9a/GLP small molecular inhibitor, better retain stem cell-like phenotypes and function during in vitro expansion. This expansion effect was further enhanced by co-treatment of SR1, an aryl hydrocarbon receptor (AHR) inhibitor. Moreover, UNC0638 treated HSPCs preferentially gave rise to megakaryocytes over other myeloid lineages when differentiating. These results suggest that G9a/GLP activity facilitates human HSPC lineage commitment and inform clinical manipulation of donor-derived HSPCs in vitro. I also applied a high-throughput shRNA screening approach to identify genes controlling early myeloid differentiation of human HSPC. To aid in identification of candidate screen hits, data from the shRNA screen were compared to known hematopoietic/hematopoietic malignancy functions and mouse HSC eQTLs. Potential self-renewal and differentiation genes were validated by a secondary screen. Further functional validations will involve gene knockdown experiments followed by flow analysis, colony-forming unit assays, and in vivo experiments in a canine transplantation model. Ultimately our results will facilitate in vitro and in vivo manipulations of human HSPC to control lineage commitment.