Freund, J.-N. | bioRxiv Science

bioRxiv2023

CD32 allows capturing blood cells emergence in slow motion during human embryonic development

During development, in the embryo proper blood cells emerge from a subset of specialized endothelial cells, named hemogenic endothelial cells (HECs), via a process known as endothelial-to-hematopoietic transition (EHT) driven by time-specific Notch signaling activation1. HECs represent an elusive cell population as they are rare and transient, rapidly generating blood cells, and specific markers are lacking. Therefore, it remains unclear how and when the hematopoietic fate is specified and how blood cell emergence is molecularly regulated. Notably, thorough characterization of this process is essential to guide the generation of therapeutic blood products in vitro from human pluripotent stem cells (hPSCs). To identify specific human HEC markers, we performed transcriptomic analysis of 28-32-day human embryos, a developmental stage characterized by active hematopoiesis. We observed that the expression of FCGR2B, encoding for the Fc receptor CD32, is highly enriched in the ACE+CD34+ endothelial cell population that contains HECs. Functional ex vivo analyses confirmed that multilineage hematopoietic potential is highly enriched in CD32+ endothelial cells isolated from human embryos. In addition, clonal analysis revealed that 90% of CD32+ hPSC-derived endothelial cells are bona fide HECs. We leveraged this specificity to study how HECs commit to the blood fate. Remarkably, our analyses indicated that HECs progress through different states culminating with the one identified by CD32 expression. Indeed CD32+ HECs no longer require Notch to generate hematopoietic progeny and display full commitment to hematopoiesis even before the expression of hematopoietic markers. These findings provide a precise method for isolating HECs primed to the blood fate from human embryos and hPSC cultures, thus allowing the efficient generation of hematopoietic cells in vitro.

developmental biology↗

bioRxiv2019

Actomyosin, vimentin and LINC complex pull on osteosarcoma nuclei to deform on micropillar topography

Cell deformation occurs in many critical biological processes, including cell extravasation during immune response and cancer metastasis. These cells deform the nucleus, its largest and stiffest organelle, while passing through narrow constrictions in vivo and the underlying mechanisms still remain elusive. It is unclear which biochemical actors are responsible and whether the nucleus is pushed or pulled (or both) during deformation. Herein we use an easily-tunable poly-L-lactic acid micropillar topography, mimicking in vivo constrictions to determine the mechanisms responsible for nucleus deformation. Using biochemical tools, we determine that actomyosin contractility, vimentin and nucleo-cytoskeletal connections play essential roles in nuclear deformation, but not A-type lamins. We chemically tune the adhesiveness of the micropillars to show that pulling forces are predominantly responsible for the deformation of the nucleus. We confirm these results using an in silico cell model and propose a comprehensive mechanism for cellular and nuclear deformation during confinement. These results indicate that microstructured biomaterials are extremely versatile tools to understand how forces are exerted in biological systems and can be useful to dissect and mimic complex in vivo behaviour.

cell biology↗