Dissecting human haematopoietic progenitors

Human haematopoiesis resembles a complex hierarchy, however most intermediate stages are only poorly defined. Efforts to characterise human progenitors have been inconsistent and failed to integrate previous knowledge. Furthermore, characterisation of normal progenitors has important implications in acute myeloid leukaemia (AML) biology. We previously established that leukaemic stem cells (LSCs) resemble the immunophenotypic progenitor compartments more closely than the stem cell fraction. Therefore, I set out to characterise human stem and progenitor cells (HSCPs) on phenotypic, molecular and functional level to complete the picture of human haematopoiesis. I purified HSPCs based on their immunophenotype from adult bone marrow (BM) and umbilical cord blood (CB) to investigate steady state and neonatal haematopoiesis. To define differentiation potentials, HSPCs were subjected to functional in vitro assays on bulk and clonal level. Limit dilution assays were used to determine the frequency of cells with multiple differentiation potentials. RNA sequencing revealed underlying lineage priming and specific gene expression signatures. I successfully characterized the incompletely defined Lin-CD34+CD38-CD45RA+ fraction in BM and CB, containing a CD10lo lymphoid-primed multipotent progenitor (LMPP) with T cell, B cell, NK cell, granulocytic and monocytic differentiation potential, and succeeded in placing it in the haematopoietic hierarchy with relation to similar lympho-myeloid progenitors defined by other groups. This research lays the foundation to characterise early human progenitors with a comprehensive toolkit on a phenotypic, molecular and functional level. Findings from this thesis might provide knowledge about potential targets in LSCs.</p

Dissecting human haematopoietic progenitors

Human haematopoiesis resembles a complex hierarchy, however most intermediate stages are only poorly defined. Efforts to characterise human progenitors have been inconsistent and failed to integrate previous knowledge. Furthermore, characterisation of normal progenitors has important implications in acute myeloid leukaemia (AML) biology. We previously established that leukaemic stem cells (LSCs) resemble the immunophenotypic progenitor compartments more closely than the stem cell fraction. Therefore, I set out to characterise human stem and progenitor cells (HSCPs) on phenotypic, molecular and functional level to complete the picture of human haematopoiesis. I purified HSPCs based on their immunophenotype from adult bone marrow (BM) and umbilical cord blood (CB) to investigate steady state and neonatal haematopoiesis. To define differentiation potentials, HSPCs were subjected to functional in vitro assays on bulk and clonal level. Limit dilution assays were used to determine the frequency of cells with multiple differentiation potentials. RNA sequencing revealed underlying lineage priming and specific gene expression signatures. I successfully characterized the incompletely defined Lin-CD34+CD38-CD45RA+ fraction in BM and CB, containing a CD10lo lymphoid-primed multipotent progenitor (LMPP) with T cell, B cell, NK cell, granulocytic and monocytic differentiation potential, and succeeded in placing it in the haematopoietic hierarchy with relation to similar lympho-myeloid progenitors defined by other groups. This research lays the foundation to characterise early human progenitors with a comprehensive toolkit on a phenotypic, molecular and functional level. Findings from this thesis might provide knowledge about potential targets in LSCs.This thesis is not currently available via ORA

Single-cell analysis reveals the continuum of human lympho-myeloid progenitor cells.

The hierarchy of human hemopoietic progenitor cells that produce lymphoid and granulocytic-monocytic (myeloid) lineages is unclear. Multiple progenitor populations produce lymphoid and myeloid cells, but they remain incompletely characterized. Here we demonstrated that lympho-myeloid progenitor populations in cord blood - lymphoid-primed multi-potential progenitors (LMPPs), granulocyte-macrophage progenitors (GMPs) and multi-lymphoid progenitors (MLPs) - were functionally and transcriptionally distinct and heterogeneous at the clonal level, with progenitors of many different functional potentials present. Although most progenitors had the potential to develop into only one mature cell type ('uni-lineage potential'), bi- and rarer multi-lineage progenitors were present among LMPPs, GMPs and MLPs. Those findings, coupled with single-cell expression analyses, suggest that a continuum of progenitors execute lymphoid and myeloid differentiation, rather than only uni-lineage progenitors' being present downstream of stem cells.MRC (MHU Award G1000729, MRC Disease Team Award 4050189188), CRUK (Program Grant to PV C7893/A12796, CRUK program grant to BG C1163/A21762), Bloodwise (Specialist Program 13001 and Project grant to 12019), an MRC PhD studentship (F.H. & Z.A.), The MRC Single Cell Award (MR/M00919X/1) to the WIMM and the Oxford Partnership Comprehensive Biomedical Research Centre (NIHR BRC Funding scheme). We thank the High-Throughput Genomics Group at the Wellcome Trust Centre for Human Genetics (funded by Wellcome Trust grant reference 090532/Z/09/Z) for generation of sequencing data. R.M. was supported by National Institutes of Health grants R01CA188055 and U01HL099999, New York Stem Cell Foundation Robertson Investigator and Leukemia and Lymphoma Society Scholar Award. A.R. was supported by an Erwin-Schroedinger Research fellowship from the Austrian Science Fund (FWF)