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  • br Experimental Procedures br Acknowledgments br Introductio

    2018-10-22


    Experimental Procedures
    Acknowledgments
    Introduction Hematopoietic stem mu opioid receptor antagonist (HSCs) reside at the apex of the hematopoietic hierarchy and generate the entire repertoire of highly specialized hematopoietic effector cells by differentiating through a succession of increasingly committed progenitors. HSCs are the only hematopoietic cell type that can differentiate into all blood lineages and self-renew for life. These properties, along with HSCs’ remarkable ability to engraft conditioned recipients upon intravenous transplantation, have established the clinical paradigm for the application of stem cells in regenerative medicine. Indeed, HSC transplantation is routinely used to treat a variety of hematological conditions, including leukemia, multiple myeloma, severe combined immunodeficiency, and myelodysplastic syndrome. Nonetheless, HSC transplantation remains a relative high-risk procedure, with the most significant factor contributing to the success of the procedure being the size of the transplanted graft (Siena et al., 2000). Enormous efforts have therefore been mounted to develop methods for expanding HSCs ex vivo, although these efforts have not yet translated to the clinic. A greater understanding of the molecular mechanisms underlying HSC fate and function will undoubtedly inform strategies for the therapeutic manipulation of these cells, and may also improve our understanding of hematopoietic malignancies derived from stem cells. The ability to purify HSCs to near homogeneity opens the door for their precise molecular characterization by microarray analysis. This approach is particularly useful for studying HSCs because it allows for the simultaneous, quantitative detection of entire transcriptomes from these rare cells. Although prior microarray studies have provided useful insights into HSC biology (Chambers et al., 2007; Forsberg et al., 2005, 2010; Rossi et al., 2005), it has proved challenging to cross-analyze data due to differences in experimental designs and technical methodologies. The ImmGen Project overcomes many of these limitations by generating transcriptome data from stem cells, defined progenitors, and various effector cells, using unified protocols of cell sorting, RNA extraction, unamplified sample preparation, and a common facility for microarray processing (Heng and Painter, 2008; Painter et al., 2011). Additional advantages of the ImmGen approach include a wider breadth of assayed hematopoietic cell types and states (∼250), increased statistical power through array number (∼700 total), and utilization of the Affymetrix GeneChip Mouse Gene ST 1.0 microarray platform, which includes probes for >24,500 coding and >1,300 noncoding transcripts. Here, we used the breadth of the ImmGen data set to delineate genes and regulators of the primitive hematopoietic cells, bringing to light conceptual advances at three levels of resolution: (1) hematopoietic stem and progenitor cells (HSPCs), (2) multipotent stem and progenitor cells, and (3) HSCs. All HSPCs showed enriched expression of metabolic growth- and proliferation-associated genes, which paradoxically were also expressed in quiescent HSCs. Genes encoding transcription factors, including a group of Kruppel-associated box (KRAB) domain-containing CH3 zinc-finger proteins that are predicted to function as transcriptional repressors, were enriched in multipotent progenitors (MPPs) and HSCs. Exposure to clinically relevant mobilizing stimuli led to alterations in the expression of HSPC regulators, as well as membrane and extracellular matrix proteins and proteases. Proximal promoter analysis of genes identified in steady-state HSPCs and mobilized HSPCs (moHSPCs) revealed enrichment of motifs representing putative binding sites for both known and unknown stem cell regulators, and ImmGen module analysis of HSC-enriched genes independently identified potential regulators. Enforced expression of one putative regulator, Hlf, resulted in robust induction of a primitive immunophenotype, sustained colony-formation activity, and enhanced self-renewal in a number of progenitor subsets ex vivo.