Molecular Events Defining Human Clonal Hematopoiesis at Single Cell Resolution

For decades, hematopoiesis has been described as a cellular hierarchy maintained by self-renewing hematopoietic stem cells (HSCs) that proceed through a series of multipotent, oligopotent and then unipotent progenitors to make blood. This can be regarded as the standard "textbook" model that is widely used as the basis to interpret molecular regulatory processes, disease processes, and develop therapeutic approaches. The presence of oligopotent intermediates is crucial to the model since they define the path from multipotent cells to unipotent cells. Common myeloid progenitors (CMPs) represent the critical oligopotent progenitor from which all My (defined as granulocyte/monocyte), erythroid (Er), and megakaryocyte (Mk) cells arise. Although the standard model is still used extensively as an operational paradigm, further cell purification and functional clonal assays have led to key revisions of the model. Through the development of more efficient assays to monitor My and lymphoid fates in single cell stromal assays and an improved sorting scheme, we previously identified human multilymphoid progenitors (MLP) as the earliest lymphoid differentiation precursor with concomitant lymphoid (T, B, NK) and myelomonocytic potential, rather than common lymphoid progenitors (CLP) (Doulatov et al, Nature Immunology 2010). Murine studies came to the same conclusion. These results raise the question of whether the opposite arm of this lineage split (My, Er, and Mk fate) is also more complex than previously thought. Indeed strong evidence has emerged from murine studies for revision to the view of murine My-Mk-Er lineage development. Until recently considerable uncertainty remained concerning the myelo-erythroid branch of human hematopoiesis. Through a novel cell-sorting scheme and a more sensitive assay to assess multilineage My, Er, and Mk fate potential contemporaneously from single cells we have provided a new framework to understand normal and disease states of human hematopoiesis (Notta et al Science 2016). These studies suggest that oligopotent progenitors are a negligible component of the human hematopoietic hierarchy of adult bone marrow. Rather, multipotent cells differentiate into unipotent cells of the My-Er-Mk lineages directly support the concept of a 'two-tier' human blood hierarchy composed of two-tiers: a top-tier containing multipotent cells such as HSCs and MPPs, and a bottom-tier composed of progenitors committed directly to My, Er, or Mk lineages. Second, the blood hierarchy is not identical across development. In human fetal liver (FL), oligopotent progenitors with My-Er-Mk and Er-Mk activity were a prominent component of the hierarchy. By contrast, the adult bone marrow (BM) was dominated by unilineage progenitors with primarily My or Er potential. Finally, our study has documented multiple origins of where Mk cells arise. We found that Mk branching differs in FL and BM. In FL, Mk progenitors were enriched, but not restricted, to the stem cell compartment; while in BM, Mk fate was closely tied to multipotent cells. This result fits the two-tier model of adult hematopoiesis since branching of Mk occurs in the top tier, at the level of HSC/MPPs. To gain a deeper understanding of the molecular basis for the two tier hierarchy, we have undertaken low cell input RNA sequencing, Enhanced Reduced Representation Bisulfite Sequencing (ERRBS), and ATAC-seq to provide a comprehensive transcriptional and epigenetic roadmap of human HSPC across development. References:Doulatov, S., Notta, F., Eppert, K. et al.Revised map of the human progenitor hierarchy shows the origin of macrophages and dendritic cells in early lymphoid development. Nature Immunology. 2010. 11, 585-593. 2. Notta, F., Zandi, S., Takayama N., et al. Distinct routes of lineage development reshape the human blood hierarchy across ontogeny. Science. 2016. 351, 176- 184. Disclosures No relevant conflicts of interest to declare.

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