Identification of the niche and phenotype of the first human hematopoietic stem cells

Abstract

In various vertebrate species, the dorsal aorta (Ao) is the site of specification of adult hematopoietic stem cells (HSCs). It has been observed that the upregulation of essential hematopoietic transcription factors and the formation of specific intra-aortic hematopoietic cell clusters occur predominantly in the ventral domain of the Ao (AoV). In the mouse, the first HSCs emerge in the AoV. Here, we demonstrate that in the human embryo the first definitive HSCs also emerge asymmetrically and are localized to the AoV, which thus identifies a functional niche for developing human HSCs. Using magnetic cell separation and xenotransplantations, we show that the first human HSCs are CD34(+)VE-cadherin(+)CD45(+)C-KIT(+)THY-1(+)Endoglin(+)RUNX1(+)CD38(-/lo)CD45RA(-). This population harbors practically all committed hematopoietic progenitors and is underrepresented in the dorsal domain of the Ao (AoD) and urogenital ridges (UGRs). The present study provides a foundation for analysis of molecular mechanisms underpinning embryonic specification of human HSCs.

Figure 1

Dorsoventral Polarization of Hematopoietic Activity in the Human AGM Region (A–C) AGM regions from five CS 14–17 human embryos were dissected into AoV, AoD, and UGRs (Figure S1), and single-cell suspensions were prepared from these tissues. CD45⁺ and CD34⁺CD45⁺ cell numbers per e.e. were assessed by flow cytometry. CFU-C numbers per e.e. were evaluated using methylcellulose culture. Tissues from the same embryo are represented by the same symbol on the plots. Mean values ± SD are shown. (D) CD34, VE-cadherin, and CD45 expression in cells from the AoV, AoD, and UGRs. VE-cadherin⁺ cells are overlaid and shown in red.

Figure 2

Phenotypic Characterization of Human AGM Region Cells (A) CD34, C-KIT, THY-1, endoglin, RUNX1, CD38, and CD45RA expression in the VE-cadherin⁺CD45⁺ cell population. The dotted lines indicate fluorescence-minus-one controls. At least two independent experiments were performed for each antigen. (B and C) Four cell populations were sorted from the total AGM region based on the expression of VE-cadherin and CD45 antigens, and plated in the CFU-C assay. Four independent experiments were performed. Mean values ± SD are shown. See also Table S1.