Recent Advances in Developmental Hematopoiesis: Diving Deeper With New Technologies

Abstract

The journey of a hematopoietic stem cell (HSC) involves the passage through successive anatomical sites where HSCs are in direct contact with their surrounding microenvironment, also known as niche. These spatial and temporal cellular interactions throughout development are required for the acquisition of stem cell properties, and for maintaining the HSC pool through balancing self-renewal, quiescence and lineage commitment. Understanding the context and consequences of these interactions will be imperative for our understanding of HSC biology and will lead to the improvement of in vitro production of HSCs for clinical purposes. The aorta-gonad-mesonephros (AGM) region is in this light of particular interest since this is the cradle of HSC emergence during the embryonic development of all vertebrate species. In this review, we will focus on the developmental origin of HSCs and will discuss the novel technological approaches and recent progress made to identify the cellular composition of the HSC supportive niche and the underlying molecular events occurring in the AGM region.

Keywords: aorta-gonad-mesonephros; embryo; hematopoietic stem cells; hemogenic endothelium; microenvironment; niche; single cell RNA sequencing; tomography sequencing.

Figure 1

Spatial and temporal locations of IAHCs and HSPCs in the aorta of zebrafish, chicken, mouse and human embryos. The endothelial to hematopoietic transition (EHT) leads to the production of single HSPCs in the aorta of zebrafish embryos, or clusters (intra-aortic hematopoietic clusters or IAHCs). IAHCs emerge exclusively in the ventral side of the aorta in chicken and human embryos (arrow heads). In contrast, IAHCs also emerge in the dorsal side of the aorta in mouse embryos (arrows). In chicken, IAHC cells ingress underneath the ventral aortic endothelium to form sub-aortic patches (SAPs). The starting time of IAHCs and HSPCs/HSCs detected is indicated for each embryo species. D, dorsal; V, ventral.

Figure 2

(A–F) Experimental approaches of key studies to identify the HSPC supportive landscape of the embryonic aorta, i.e. the studies of Charbord et al. (135) (A), Mascarenhas et al. (93) (B), McGarvey et al. (136) (C), Crosse et al. (117) (D), Yvernogeau et al. (86) (E) and Xue et al. (137) (F). The embryo species and stages, the type of cells/tissues analyzed, and the experimental approach used for each study are indicated. The interactives website resources provided in each study are also indicated (in blue). AGM, Aorta-Gonad-Mesonephros; FL, Fetal liver; BM, Bone marrow; E, Embryonic day; IAHCs, Intra-Aortic Hematopoietic Clusters, HSCs, Hematopoietic stem cells; Nc, Notochord; Ao, Aorta; BM, Bone marrow; CS, Carnegie stage; CHT, Caudal hematopoietic tissue; hpf, hour post-fertilization.

Figure 3

Schematic illustration of single cell sequencing of tissue slices and high-multiplex protein detection on tissue sections. (A) Schematic representation of 10X Genomics Visium workflow. Fresh-frozen or paraffin embedded tissue sections are prepared on specialized slides that contain several capture areas or grid. Each grid position has a known unique barcode, which is used for reconstructing the tissue after sequencing. These tissues can be stained with fluorescent antibodies or with one of the principle tissue stains, e.g. hematoxylin & eosin, and subsequently imaged by microscopy prior RNA capture. Cells are then permeabilized and the RNA content is captured for barcoding and library preparation. The library is sequenced by standard procedures and the data can be visualized in relation to the imaged tissues. (B). The use of multiple fluorescent proteins and quantum dots to simultaneously label different cell components is limited to the ability to resolve spectral overlap between the different fluorochromes. To increase the number of labels, classic fluorescent proteins have been replaced by specific elements or isotopes such as noble and post-transition metals, rare-earth elements and halogens, which can be detected by mass cytometry. Antibodies against specific proteins (e.g. receptors or transcription factors) can be tagged with these isotopes and used for staining of tissue sections and an image of the general morphology can be taken. A UV-laser evaporates the cells in a typical confocal scanning movement (X,Y) and fumes of the cells are transported by an inert gas into the mass cytometer for measurement. The measured isotopes and the corresponding X,Y coordinates can then be used to reconstruct an image which can be superimposed on top of the microscope images.