Understanding the heterogeneity of the hematopoietic stem cells

The hematopoietic system is replenished and maintained throughout life by rare hematopoietic stem cells (HSCs) that reside in the bone marrow (BM) of adult mammals. Over the last 20 years, the advancement in the field lead to the acknowledgement of the heterogeneity within the HSC compartment unraveling the presence of HSC subsets with certain mature blood lineage preferences so called lineage-biased (Li-bi) HSCs. Studying the heterogeneity and lineage bias within the HSC compartment is crucial not only to understand the functional and molecular mechanisms behind this lineage skewing but can also shed light on the emergence of hematological malignancies subsequently paving the way to find new therapeutic targets, better treatment options and more selective alternatives of BM transplantation. Recent developments have taken advantage of immunophenotypic markers for prospective isolation of cells. The cell surface markers can be used to enrich for HSCs but cannot purify. Current markers cannot resolve heterogeneity within the HSC compartment, highlighting the importance of continuing efforts on identifying new cell surface markers that enrich Li-bi HSC subtypes. In paper I, we demonstrate that CD49b cell surface marker subfractionates the most primitive HSC compartment into two; CD49b– HSCs with myeloid bias, high self-renewal potential and the most quiescent state, and CD49b+ HSCs with lymphoid bias, lowered selfrenewal potential and more proliferative state. Furthermore, we show that both subsets have similar transcriptome profiles but distinct epigenetic landscapes highlighting that the lineage-bias is regulated via epigenetic mechanisms. In paper III, we show that using the additional cell surface marker CD229, the remaining heterogeneity within the CD49b+ HSCs can be resolved into two functional subsets as CD49b+CD229– and CD49b+CD229+. The CD49b+CD229– fraction shows long-term and stable reconstitution and the CD49b+CD229+ fraction enriches for multipotent progenitor cells having short term activity. Hematopoietic aging is associated with myeloid skewing, delayed, and reduced immune response and higher incidences of myeloid malignancies. The composition of HSC compartment changes with a shift toward an increased proportion of myeloid biased HSCs in elderly both in human and mice. However, the molecular mechanisms behind this phenomenon are not completely understood. In paper II, we show that the CD49b– HSC maintains its myeloid bias in the peripheral blood of the young, young adult and old age groups whereas the CD49b+ HSC shifts from lymphoid bias in young and young adult to lineage-balance (no bias) in aged mice. In addition, we demonstrate that both subsets are equally active in young and have similar chromatin landscapes with different levels of accessible regions in old mice. The B cell lineage priming occurs downstream of HSCs starting at the branching point of multipotent progenitors in the hematopoietic hierarchy. The B cell development is highly regulated by transcriptional factors. In paper IV, we show that combined loss of transcription factors FOXO1 and FOXO3 prevents the B cell development by blocking it at the BLP stage. Moreover, we demonstrate that FOXO3 plays a crucial role in regulating the B cell lineage priming higher up in the hematopoietic hierarchy already as early as the LMPP level. Collectively, this thesis identifies cell surface markers that resolves the functional heterogeneity of the HSCs, gives insights into how the lineage bias is regulated during aging, and unravels the effect of transcription factors in B cell development

Bcl11a Deficiency Leads to Hematopoietic Stem Cell Defects with an Aging-like Phenotype

SummaryB cell CLL/lymphoma 11A (BCL11A) is a transcription factor and regulator of hemoglobin switching that has emerged as a promising therapeutic target for sickle cell disease and thalassemia. In the hematopoietic system, BCL11A is required for B lymphopoiesis, yet its role in other hematopoietic cells, especially hematopoietic stem cells (HSCs) remains elusive. The extensive expression of BCL11A in hematopoiesis implicates context-dependent roles, highlighting the importance of fully characterizing its function as part of ongoing efforts for stem cell therapy and regenerative medicine. Here, we demonstrate that BCL11A is indispensable for normal HSC function. Bcl11a deficiency results in HSC defects, typically observed in the aging hematopoietic system. We find that downregulation of cyclin-dependent kinase 6 (Cdk6), and the ensuing cell-cycle delay, correlate with HSC dysfunction. Our studies define a mechanism for BCL11A in regulation of HSC function and have important implications for the design of therapeutic approaches to targeting BCL11A

CD49b identifies functionally and epigenetically distinct subsets of lineage-biased hematopoietic stem cells

Hematopoiesis is maintained by functionally diverse lineage-biased hematopoietic stem cells (HSCs). The functional significance of HSC heterogeneity and the regulatory mechanisms underlying lineage bias are not well understood. However, absolute purification of HSC subtypes with a pre-determined behavior remains challenging, highlighting the importance of continued efforts toward prospective isolation of homogeneous HSC subsets. In this study, we demonstrate that CD49b subdivides the most primitive HSC compartment into functionally distinct subtypes: CD49b(-) HSCs are highly enriched for myeloid-biased and the most durable cells, while CD49b(+) HSCs are enriched for multipotent cells with lymphoid bias and reduced self-renewal ability. We further demonstrate considerable transcriptional similarities between CD49b(-) and CD49b(+) HSCs but distinct differences in chromatin accessibility. Our studies highlight the diversity of HSC functional behaviors and provide insights into the molecular regulation of HSC heterogeneity through transcriptional and epigenetic mechanisms

FOXO Dictates Initiation of B Cell Development and Myeloid Restriction in Common Lymphoid Progenitors

The development of B cells relies on an intricate network of transcription factors critical for developmental progression and lineage commitment. In the B cell developmental trajectory, a temporal switch from predominant Foxo3 to Foxo1 expression occurs at the CLP stage. Utilizing VAV-iCre mediated conditional deletion, we found that the loss of FOXO3 impaired B cell development from LMPP down to B cell precursors, while the loss of FOXO1 impaired B cell commitment and resulted in a complete developmental block at the CD25 negative proB cell stage. Strikingly, the combined loss of FOXO1 and FOXO3 resulted in the failure to restrict the myeloid potential of CLPs and the complete loss of the B cell lineage. This is underpinned by the failure to enforce the early B-lineage gene regulatory circuitry upon a predominantly pre-established open chromatin landscape. Altogether, this demonstrates that FOXO3 and FOXO1 cooperatively govern early lineage restriction and initiation of B-lineage commitment in CLPs