In-Depth Proteomic Characterization of Lineage-Biased Hematopoietic Progenitor Cells in the Fetus and Adult

The process of hematopoiesis is subject to extensive ontogenic remodeling that is accompanied by alterations in cellular fate both during normal development and upon malignant transformation. Although the functional differences between fetal and adult hematopoiesis are well established, the responsible molecular mechanisms have long remained largely unexplored at the proteomic level. We hypothesize that an intrinsically programmed proteomic switch in hematopoietic stem and progenitor cells (HSPCs) during ontogeny regulates the outcome of hematopoiesis both during normal development and upon leukemia initiation, and that the proteomic makeup of the leukemia-initiating cell has an instructive role in determining the outcome of the resulting cancer.

In our latest work, we utilized quantitative mass spectrometry-based proteomics to characterize and compare the proteomic makeup of fetal and adult Lin^- Sca-1⁺ cKit⁺ (LSK) HSPCs (Jassinskaja et al., 2017, Cell Reports), representing all of the earliest stem and progenitors in fetal and adult hematopoiesis. We identified differences in several important cellular processes not previously described to play a role in hematopoiesis, highlighting the need for applying proteomic-centric approaches in the field. In order to further increase our understanding of normal and malignant hematopoiesis during ontogeny, we are now continuing this work by focusing on more stringently defined populations of lineage-biased hematopoietic progenitor cells (HPCs).

Here, we have utilized encapsulated methods for preparation of microscale samples in combination with state-of-the-art mass spectrometry to gain deep coverage of the proteome of 100,000 fetal (E14.5) and adult lymphoid-primed multipotent progenitors (LMPPs), common lymphoid progenitors (CLPs) and granulocyte-macrophage progenitors (GMPs). Our analysis resulted in the identification and quantification of 4189 proteins, with over 200 proteins per cell type displaying differential expression between the fetus and the adult. Importantly, the differentially expressed proteins were enriched for a broad variety of biological processes. Similar to our previous findings in HSPCs, for all three cell types, proteins higher expressed in the fetus showed a strong enrichment for cell cycle- and translation-related processes, whereas those higher expressed in the adult were enriched for processes related to immune response and redox homeostasis. Our preliminary analysis of hematopoietic cell subset signatures associated with the differentially expressed proteins suggests a stronger lymphoid bias in fetal compared to adult LMPPs as well as CLPs. Surprisingly, the proteomic signature of fetal GMPs suggests a retained megakaryocyte-erythroid potential, which is corroborated by a significantly higher expression of megakaryocyte progenitor marker CD41 on the fetal cells. Upon analyzing expression of transcription factors (TFs) in fetal and adult HPCs, we could confirm differential expression of TFs known to have ontogeny-specific roles in hematopoiesis (e.g. Arid3a and Etv6). Importantly, we also identified several differentially expressed TFs that could represent novel regulators of fetal- and adult-specific features of hematopoiesis, such as Irf8, Btf3, Mndal and Pura. Furthermore, the difference in expression of Irf8 observed here could indicate a previously unknown ontogenic switch in the balance between neutrophil and monocyte production from myeloid-competent progenitors. Lastly, our data shows strong indications of a differential sensitivity towards Rho kinase inhibition between the fetal and the adult HPCs.

Collectively, our work represents a significant advancement in the understanding of the molecular programs that govern ontogenic differences in hematopoiesis and provides a solid foundation for future investigation of which factors are responsible for the difference in susceptibility and outcome of different leukemias in infants and in adults.