Dissertation

Multi-Disciplinary Approaches to Investigate Hematopoiesis

Song, Baobao Annie

2023

Overview

Hematopoiesis is a complex and tightly regulated biological system in which the stem cells and progenitors balance between mature lineage outputs and self-renewal. Environmental stimuli, genetic predispositions, and host responses together control and alter underlying transcriptional network. Various animal models and single-cell multi-omics approaches have been developed to unravel the network and mechanisms associated with it. This dissertation characterizes relationship between gut microbiome (environmental stimuli) and host; describes human and murine hematopoietic stem cells and progenitors; and demonstrates methods to incorporate with biological models to derive insightful biological readouts.Microbiome models have informed our understanding of microbiome-dependent metabolites, their role in homeostatic hematopoiesis, and nominated some metabolites as potential therapeutics. However, conclusions about gut microbiota-induced biological functions have been called into question after recent results from metabolite clinical trials and conflicting findings across studies. To systematically evaluate the impact of durably different gut microbiome on homeostatic hematopoiesis, we analyzed three C57BL/6 mouse microbiome models with defined flora, and contrasted with germ-free and standard-pathogen-free C57BL/6 mice. Metagenomics and metabolomics confirmed distinct microbial species present, their relative diversity, and associated changes in physiology and serum metabolites. We find that a minimal microbiome composition restores the majority of metabolites absent in germ-free mice, but community complexity controls metabolite abundance and perturbs metabolite pathways and transcriptional signatures. However, immunophenotyping reveals comparable innate and adaptive immune populations across models. Bone marrow transplant reveals normal homeostatic HSC function, and in vitro assays reveal similar neutrophil innate immune function across models. Thus, defined flora microbiome models clarify the impact of live bacterial signals in controlling homeostatic hematopoietic numbers and function.Advancements in single-cell technologies, like CITE-seq, have enhanced our understanding of blood cell development. However, translating CITE-seq findings to flow cytometry faces challenges. We conducted molecular titration of 266 antibodies on human primary bone marrow cells, identifying 132 relevant antibodies that inform an atlas of 89 clusters, bridging flow cytometry and CITE-seq. We integrated high-dimensional flow cytometry and CITE-seq to evaluate 132 surface markers across donors and technologies, concluding with 39 consistently expressed markers and innovative isolation strategies for neutrophil precursors and megakaryocyte/erythrocyte progenitors.Hematopoietic stem cells (HSCs) are functionally heterogeneous, but molecular drivers of their functionality remain unclear. A gene regulatory network (GRN) centric to HSC quiescence was identified by in vivo activation, and molecular profiles of HSC subsets were compared in two reporter systems to establish hierarchically organized HSC states. Tox, a regulator of T cell development, was found essential for HSC quiescence. Loss of Tox led to worse transplant outcomes and perturbed key TFs regulating HSCs. . Collectively, these synergistic thesis projects nominate new stem and progenitor cell populations, novel isolation and functional characterization strategies and underlying gene regulatory networks in normal and microbiome-impacted hematopoiesis.

• Publisher: ProQuest Dissertations & Theses
• Subject: Bioengineering / Cellular biology / Immunology / Molecular biology / Physiology
• ISBN: 9798346853442