Megakaryocyte heterogeneity at the bone marrow sinus wall: interactions with pericytes and their implications on platelet biogenesis and function

• Applicant: Professor Dr. Harald Schulze
• Subject Area: Immunology ; Cell Biology
• Term: since 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 535197976

All blood cells derive from hematopoietic stem cells (HSCs), located in the bone marrow (BM). Mature progenitor cells are found adjacent to the blood vessels in the marrow, before they finally cross the sinus wall and are shed into the circulation. Blood platelets are small cellular fragments devoid of a nucleus and essential for hemostasis. They are formed by megakaryocytes (MKs), large cells that release up to 1000 platelets into the blood stream. While thrombopoiesis is well understood in vitro, it remains unclear how it is regulated at the BM vascular niche. Mature MKs polarize and start to form long protrusions (proplatelets) through pores across the endothelial layer. During this process, they encounter matrix proteins like collagens or laminins without getting activated. Recently, we identified CXCL12-abundant reticular (CAR) cells that sheath BM sinusoids as a cellular component which seem to be retracted at the sites were proplatelet formation takes place, implying that CAR cells contribute in regulating platelet biogenesis. In this research proposal we aim to address how the communication between MKs and CAR cells occurs at the BM sinus wall and how thrombopoiesis is regulated under homeostatic and inflammatory conditions: (1) The proposed research project aims to characterize the interface between MKs and CAR cells in situ in mouse femur sections, using confocal laser scanning microscopy. We will apply a newly developed thick sectioning method which allows us to record information in three dimensions rather than on 2D sections. (2) We will further analyze putative receptor-ligand pairs between MKs and CAR cells (including CXCL12-CXCR4/7; PDGF-PDGFR; VCAM-1/VLA-4) to address how the mutual communication is mediated. We will apply our knowledge also to intravital two-photon microscopy with reporter mouse strains, which will allow us to visualize thrombopoiesis within the BM. We will interfere with these interactions using blocking antibodies, small molecules, lentiviral knock-down and CRISPR/Cas9-based approaches using stromal cell lines MS-5 and ST-2 and finally primary BM-derived CAR cells. (3) As MKs show a significant heterogeneity based on their transcriptomic signature, we aim to decipher distinct MKs and CAR cells based on their location within the BM, complemented by approaches of spatial resolution using single cell RNAsequencing techniques. (4) HSC transplantation (HSCT) requires total body irradiation (TBI) prior to transfusing the donor-derived stem cells. TBI leads to a fundamental relocation of CAR cells and to ectopic platelet release into the BM cavity. We will also study how the newly formed platelets show an altered platelet function. This might affect the way how thrombotic events despite overall low platelet counts could be explained after HSCT. We expect that this proposal will provide a better understanding how CAR cells provide an additional layer of regulation for platelet production across the BM sinus wall.

• DFG Programme: Research Grants