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UBC Theses and Dissertations

Characterization of the lympho-neutrophil/monocyte restriction process in human cells Wang, Fangwu


The early stages and regulation of human B lymphocyte differentiation from multipotent hematopoietic progenitors with dually restricted B plus neutrophil/monocyte (NM) differentiation potential are poorly understood. To address this challenge, unique phenotypes of cells with dual B and NM differentiation potential and their restricted progeny were first identified. Culture systems that support the restriction of human B+NM to B or NM phenotypes during their stimulated expansion were then developed and used to track, retrospectively, the division histories of all three phenotypes. The results show that the self-renewal divisions of dual potential (B+NM) progenitors have longer cell cycle transit times than those of their expanding B- and NM-restricted progeny. A proposed model of these results suggests the existence of a transient intermediate state just prior to the acquisition of phenotypes of B- and NM-restricted cells. Given the striking global reduction in the H3K27me3 modification of histones previously seen in mature monocytes compared to the B+NM progenitors, a next goal was to determine if this change occurs during an early step in the B+NM lineage restriction process. To investigate this possibility, the capacity of B+NM progenitors to generate B- and/or NM-restricted cells in vitro was examined in the presence of a specific EZH2 inhibitor of H3K27me3 histone modification. The results revealed a significant and selective inhibition of B-restricted cell outputs in the cultures containing the EZH2 inhibitor, but without effects on the outputs of NM-restricted cells. Interestingly, clonal analyses showed the inhibitor to mainly reduce the expansion of cells with the B+NM phenotype. Together, these findings implicate an active role of EZH2, and likely an earlier state of histone modifications in retaining essential properties of B+NM and their downstream B, but not NM progeny. Taken together, these results reveal important changes in cell cycle transit control and a previously unrecognized complexity and timing in the steps cells with dual B and NM potential undergo during their downstream restriction. They also provide new tools to examine in more detail the molecular events that underpin the processes of B+NM progenitor lineage restriction that are of potential relevance to many human diseases.

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