Human embryonic ACE endothelial cells express arterial and haemogenic markers.+

a, A transverse section of the AGM region of a 23 dpf (CS10, top, n = 2 independent) and a 27 dpf (CS12, bottom, n = 3 independent) human embryo, immunostained with ACE (left, in red), RUNX1 (middle, in green) and merge (right). Ao, aorta; NT, neural tube. Scale bars, 50 μm. b–e, An RNA-seq analysis of human embryonic populations isolated from four CS12–CS13 embryos, referred to as ‘donor’: E1, E2, E3 and E4. The ACEneg population is coloured in beige and ACE population in light blue. PCA (b) of the top 500 DEGs within human embryonic populations. A heatmap of DEGs within human embryonic populations (c), where gene counts were corrected for donor and the rlog gene expression values shown in rows and tiles referring to DEGs are coloured according to upregulation (red) or downregulation (blue). A heatmap of selected pan-endothelial (CD34, CDH5, PECAM and TEK), vein-specific (NR2F2 and FLRT2), arterial-specific (GJA5, DLL4, CXCR4 and HEY2) and haemogenic (MYB, GFI1 and CD44) gene expression (d), where the rlog gene expression values shown in rows and tiles referring to DEGs are coloured according to upregulation (red) or downregulation (blue). A barplot (e) showing significantly enriched GO terms (Fisher exact test; FDR <0.05) using ORA on DEGs. The barplot shows enriched terms grouped by custom categories: cell cycle (upregulated in ACE+neg), migration and adhesion (upregulated in ACE).+

During embryonic development, blood cells are derived from a distinct type of endothelial cell known as hematopoietic endothelial cells (HECs). However, HECs are rare in early embryos and present only within a narrow developmental window, complicating their distinction from regular endothelial cells. The markers currently used to identify HECs, such as CD34 and CD44, suffer from issues with specificity and sensitivity, making accurate differentiation challenging. This limitation hampers the purification and functional research of HECs. Identifying a more specific marker would significantly enhance the identification and isolation of HECs, thereby advancing related research. While previous studies have highlighted the critical role of HECs in hematopoiesis, the precise molecular mechanisms and regulatory pathways remain largely unknown. Comprehensive research into the molecular characteristics and signaling pathways of HECs will enhance our understanding of the fundamental principles of blood cell generation, providing theoretical guidance for in vitro hematopoietic cell generation. Rebecca et al. aimed to identify surface proteins that can specifically mark HECs through transcriptome analysis and to use endothelial cells derived from human embryos and human pluripotent stem cells (hPSCs) to verify the role of the identified markers in generating hematopoietic cells in vitro. Additionally, they sought to investigate the transcriptional changes of HECs at various developmental stages to elucidate the mechanisms underlying their transition to hematopoietic cells.

The research team initially collected endothelial cells from 28 to 32-day-old human embryos for transcriptome analysis and discovered that CD32 (FCGR2B) was highly enriched in these cells, particularly in HECs. This suggests that CD32 could serve as a specific marker to distinguish HECs from other endothelial cells. Next, immunofluorescence staining was performed on cross-sections of the AGM region of human embryos to analyze the colocalization of CD32, CD34, ACE, and RUNX1 markers. The results indicated that, in 27-day-old embryos, CD32 colocalized with CD34, ACE, and RUNX1, further supporting CD32 as a specific marker for HECs. Using flow cytometry to sort CD32+ and CD32neg cells from the AGM and YS regions, it was found that CD32+ cells generated significantly more colony-forming cells (CFCs) and were capable of producing various types of hematopoietic cells. This demonstrated the strong multipotent hematopoietic potential of CD32+ cells, validating CD32 as a marker for HECs. Finally, single-cell RNA sequencing of sorted CD32+ and CD32neg cells revealed that CD32+ cells had significantly upregulated expression of hematopoiesis-related genes and were enriched in genes associated with cell migration and adhesion. This finding elucidated the molecular characteristics and transcriptional changes of HECs at different developmental stages, providing crucial data for understanding the mechanisms of hematopoietic cell generation.

In summary, this study successfully validated CD32 as a specific marker for HECs. The findings from transcriptome analysis, immunofluorescence analysis, functional validation experiments, and single-cell RNA sequencing collectively underscore the critical role of CD32 in identifying and isolating HECs. This establishes a novel method for generating hematopoietic cells in vitro. Compared to traditional methods, this approach not only provides high specificity, efficiency, and diversity but also elucidates the transcriptional changes and hematopoietic mechanisms of HECs during development. These insights offer significant advancements in regenerative medicine and cell therapy.

Regrettably, this study only involved endothelial cells from 28 to 32-day-old human embryos, a relatively narrow time window that does not cover a broader range of embryonic developmental stages. Additionally, although transcriptome analysis identified key genes and signaling pathways, their specific roles in determining HEC fate remain underexplored. The absence of functional validation for these key genes makes it challenging to fully understand their roles in HEC development and hematopoiesis. Future research should employ gene-editing technologies, such as CRISPR-Cas9, to perform functional studies on these key genes, elucidating their precise roles in HEC differentiation and hematopoiesis. Furthermore, in-depth investigations of the identified signaling pathways are necessary to understand their regulatory mechanisms in HEC fate determination.

Overall, by advancing the study of HEC molecular mechanisms, optimizing in vitro culture conditions, validating long-term safety and functionality, and promoting cross-species comparisons and interdisciplinary collaborations, this field can progress significantly. Such advancements will provide a robust theoretical foundation and technical support for regenerative medicine and cell therapy.

Reference：

Scarfò, Rebecca, et al. "CD32 captures committed haemogenic endothelial cells during human embryonic development." Nature Cell Biology (2024): 1-12.