Endoglin expression level discriminates long-term hematopoietic from short-term clonogenic progenitor cells in the aorta

Abstract

CD105 is an auxiliary receptor for the transforming growth factor beta superfamily, highly expressed on proliferating endothelial cells and adult hematopoietic stem cells. Because CD105 mRNA expression was reported in the developing aortic region, we further characterized its expression profile in the aorta and examined the hematopoietic potential of CD105(+) cells. Aortic endothelial cells, intra-aortic hematopoietic cell clusters and the purified cell fraction enriched in progenitor/hematopoietic stem cell activity expressed CD105. Aortic hematopoietic short-term clonogenic progenitors were highly enriched in the CD105(intermediate) population whereas more immature long-term progenitors/hematopoietic stem cells are contained within the CD105(high) population. This places CD105 on the short list of molecules discriminating short-term versus long-term progenitors in the aorta. Furthermore, decreasing transforming growth factor beta signaling increases the number of clonogenic progenitors. This suggests that CD105 expression level defines a hierarchy among aortic hematopoietic cells allowing purification of clonogenic versus more immature hematopoietic progenitors, and that the transforming growth factor beta pathway plays a critical role in this process.

Figure 1.

Immunofluorescence analysis of CD105, CD31, CD144 and c-kit expression in the mouse (A-G) AGM before and at the time of hematopoiesis and CD105 expression in the human AGM (H). (A and C) CD105/CD31/DAPI. (B-D) CD105/CD144/DAPI. (E) CD105/c-kit. Immunohistochemistry. Cross sections. (A and B) E9 embryos. Both CD31 (A) and CD144 (B) are co-expressed with CD105 in endothelial cells, as testified with the yellow color resulting from the overlay. Bar=30 μm C, (D) E10 embryos. Coexpression is similar to that seen at E9. Bar=50 μm. (E) E10 embryo. CD105 and c-kit are co-expressed at the level of the clusters. Bar=30 μm. (F) E11 mouse embryo; cross section. CD105 immunofluorescence merged with dichroic bright field. The signal is restricted to the endothelium and to hematopoietic cells of the fetal liver and the AGM. The white arrow points to a hematopoietic cluster in the aortic floor. Bar=100 μm. Ao: aorta; C: coelom; CV: cardinal vein; FL: fetal liver; NT: neural tube; St: stomach; UA: umbilical artery. (G) E11 mouse embryo; CD105 immunofluorescence. Close view of the aorta and hematopoietic clusters. The two hematopoietic cluster-composing cells display a variable level of CD105 expression. Bar=20 μm. (H) 30 day-old human embryo; cross section; CD105 immunofluorescence merged with dichroic bright field. A variable level of expression is also visible in the hematopoietic cluster. Bar=30 μm.

Figure 2.

Endothelial and hematopoietic marker analysis of CD105 positive cells in the E11 AGM. Each pannel is the result of one representative experiment. Data in the text represent the mean of at least two to seven independent experiments. Whole E11 AGM cells were double stained with anti-CD105 and-CD34 (A), -CD144 (B), -CD45 (C), CD41 (D), and triple stained with either CD105, CD144 and CD45 (E) or CD105, c-kit and CD34 (F). Numbers indicate the percentage of positive cells in the corresponding gates.

Figure 3.

Subpopulations of CD105 expressing cells in the E11 mouse AGM and analysis of their hematopoietic potentials. (A) FACS analysis of the CD105-expressing cells in the E11 mouse AGM. A CD105^int (0.70%) and a CD105^hi (2.83%) population are clearly visible. (B) Compared clonogenic potential of the CD105 sub-populations. After sorting, the CD105^neg, CD105^pos, CD105^int and CD105^hi sub-populations were plated in methylcellulose medium. Hematopoietic colonies were scored after 14 days of culture. Data represent the mean ± SEM from 3–6 independent experiments. (C) Comparison between Day 7 and Day 35 CAFC frequency in the CD105^int and the CD105^hi sub-populations. Exp1, Exp2, Exp3 are 3 independent experiments. (D) RT-PCR analysis of the BMP/TGFβ pathway in the CD105 populations in the E11 AGM. The presence of several TGFβ-specific receptors and the strong TGFβ1 expression suggest that a TGFβ pathway is active in CD105-positive cells. Primers and size of amplification products are given in the Online Supplementary Table S1. (E) Role of a soluble form of endoglin (sEnd) on the clonogenic potential of AGM cells. A 3-day explant culture of E11 AGM was performed in the absence or in the presence of 500ng/mL of sEnd. After collagenase dissociation, cells were plated in methylcellulose medium, and hematopoietic colonies were scored after 11 days. Data represent the mean ± SEM from 3 independent experiments.