Early fate decisions in hematopoietic stem and progenitor cells. Through the lens of genomic and functional assays

Sammanfattning: Hematopoietic stem cells (HSCs) are rare cells on top of the differentiation hierarchy of hematopoiesis. HSCs are unique in their combined capacity to differentiate into all mature blood lineages and self-renew to maintain the HSC pool. Based on classical models of hematopoiesis in mouse, the self-renewal potential of HSCs is gradually and step-wise lost during the transition from long term (LT)-HSCs to multipotent progenitors (MPPs) accompanied by upregulated expression of the cell-surface marker FMS-like tyrosine kinase 3 (Flt3). The Flt3+ multipotent progenitors serve as developmental intermediates for hematopoietic lineage priming. Notably, the 25% highest Flt3+ cells, known as lymphoid-primed MPPs (LMPPs), have been defined as restricted, lymphoid primed cells with decreased megakaryocyte and erythroid (MegE) priming. However, recent single cell RNA sequencing (scRNA-seq) studies question the step-wise model of HSC differentiation and instead suggest a continuum model of the early hematopoietic hierarchy, where the first differentiation events occur in a low-primed cloud of HSPCs without sharply defined gene expression programs. In this model, no transition of different lineages from MPPs with intermediate gene expression occur, instead these progenitors are largely comprised of uni-lineage-primed cells. The overall aim of this thesis is to investigate how cellular-fate options emerge in the cloud of hematopoietic stem/progenitor cells (HSPCs), at what stage the multipotency gives way to lineage priming, and how this stage can be detected. For this aim, single-cell (sc) chromatin accessibility (ATAC-seq), scRNA-seq and sc-qPCR analysis were employed extensively to identify the transition of HSCs to lineage restricted multipotent progenitor cells and functionally validated using in vivo and in vitro assay.In paper I, scATAC-seq was used to map the accessibility of 571 transcription factor (TF)-binding motifs as a measure of lineage priming along the Flt3 differentiation axis. The resulting data identified a transition point of highly lineage-primed cells within the continuum of HSPCs where self-renewal and multipotency was lost and lineage commitment initiated. This transition point is characterized by down-regulation of CD9 and up-regulation of Flt3 cell surface expression. Within the Flt3 intermediate population (Flt3int), LSKFlt3intCD9high cells display co-incidental stem and multi-lineage primed chromatin states while the downstream LSKFlt3intCD9low contain an LMPP-like program. Also, this priming seems to initiate in the epigenome without being starkly reflected in the transcriptome. In order to validate the genomic data from the aforementioned analysis, we established in vitro culture systems to functionally examine the differentiation fates of cells at a clonal level (Paper II). The result confirms that LSKFlt3intCD9high cells generated more multilineage progeny compared to clones within the LSKFlt3intCD9low fraction. It has been shown extensive changes in heterogeneity of human hematopoietic cells with age. For example increase HSCs frequency and myeloid output while lymphoid output is decreased. However human immunophenotypic changes associated with aging have received little attention. To this end we in paper III examined CD9 cell surface expression in correlation with molecular programs and functional features of human HSPCs throughout life and in leukemia. Interestingly, only a small fraction of HSPCs expressed CD9 in neonatal hematopoiesis and in young adult bone marrow while CD9 expression substantially increased during situations of myeloid and megakaryocytic biased hematopoiesis, such as during ageing or in chronic myeloid leukemia (CML). Thus, CD9 represents an HSC marker for myeloid-biased hematopoiesis.