Characterization of purified intraembryonic hematopoietic stem cells as a tool to define their site of origin

Abstract

Little is known about hematopoietic stem cell (HSC) development from mesoderm. To gain more information on the intraembryonic HSC site of origin, we purified multipotent hematopoietic progenitors from the aorta-gonads-mesonephros (AGM) of mice. This population, expressing c-Kit, AA4.1, CD31, and CD41, but not Flk1, and mainly negative for CD45, proved capable of long-term reconstitution in sublethally irradiated Rag2gammac(-/-) recipients. We assigned the expression of GATA-2, GATA-3, and lmo2 to AGM-HSC, whereas erythromyeloid progenitors express only GATA-2. This unique combination of surface markers and transcription factors could be allocated in the AGM to the intraaortic clusters and the subaortic patches underlying aortic endothelial cells. Taken together, those data indicate that embryonic HSCs (i) differ from their fetal liver and adult counterpart by the low expression of CD45, (ii) do not colocalize with aortic endothelial cells as previously thought, and (iii) are localized, at 10.5 days postcoitum, in the splanchnic mesoderm underlying aortic endothelial cells, within GATA-3(+)CD31(+) cell clusters.

Fig. 1.

Distribution of multipotent and erythromyeloid precursors in 10.5-dpc AGM and YS. (A) AGM and YS cells at 10.5 dpc were fractionated according to the expression of CD45, c-Kit, and CD41. We could identify three fractions, A (CD45⁺), B (c-Kit^-), and C (c-Kit⁺CD45^-/lo). All c-Kit⁺ cells also express CD41. (B)In AGM and YS, fraction C was subdivided into D (c-Kit⁺AA4.1^-) and E (c-Kit⁺AA4.1⁺), shown on the plots. AGM-E cells were further fractionated with CD41; E′ represents 66% of AGM fraction E. (C) Studies in single-cell cultures and limiting dilution analysis provided the frequency of multipotent and bipotent (erythromyeloid) precursors in each fraction. Absolute numbers (frequency × number of cells) presented here are calculated according to the total number of cells in AGM and YS, estimated to be 5,000 and 50,000, respectively.

Fig. 2.

Distribution of endothelial and hematopoietic markers in cells from YS and AGM. (A) Flk1 (Center) and CD41 (Right) expression within the two gated c-Kit⁺AA4.1⁺ populations (Left), after exclusion of CD45⁺ cells. (B) CD45^-/loc-Kit⁺AA4.1⁺ and CD45^-c-Kit^- cells from AGM and YS were sorted and analyzed for expression of Lmo2, GATA-3, and GATA-2. The transcript expression is represented, after normalization to hypoxanthine phosphoribosyltransferase, by the percentage of its expression in the standard population (15-dpc fetal liver depleted of erythrocytes). Levels of expression were divided into four groups according to the percentage of expression relative to the standard. Means and standard deviations on triplicates from two individual experiments are shown. No background shading, <25%; yellow shading, 25–100%; red shading, >250%. (C) CD45 and L-plastin transcript distribution within YS and AGM fractions E and B.

Fig. 3.

Localization of AGM hematopoietic subsets in 10- to 10.5-dpc (30–35S) embryos. Confocal expression analyses (A) allow one to discriminate between macrophages (CD45⁺CD31⁺CD41^-, arrowheads) and HSCs (CD41⁺CD31⁺CD45^-/lo, arrows). HSCs are located within the intraaortic clusters (stars) and within the SAPs (asterisks). (B) CD31 is expressed by ECs and by GATA-3⁺ SAPs (asterisk). (Bars, 50 μm.)

Fig. 4.

Localization of AGM-HSCs and their environment in 10.5-dpc (35–40S) embryos. From 10.5 dpc (35–40S), the subendothelial CD31⁺GATA-3⁺ layer evolves into more compact SAPs (asterisks), which express GATA-2 mRNA (A) in addition to GATA-3 (B). Confocal analyses (C and D) show that HSCs are located within the SAPs. CD45⁺ macrophages (arrowheads) do not express CD41. XZ projection of the image stack (D) that comprises the top section in C shows that CD31⁺CD41⁺ HSCs are present only in the ventral part of the AGM (below the straight line joining the cardinal veins (CV)). Their distribution forms a continuum from the SAPs (asterisks) to the aortic floor. Note that CD31⁺CD41⁺ HSCs constitute only a minority of the CD31⁺GATA-3⁺ SAP cells. Stainings in A–C were performed on alternate sections of the same embryo. (Bar, 50 μm.)

Fig. 5.

Toward a model for emergence and migration of embryonic HSCs. (A) Localization of hemogenic-related structures, SAPs (purple), and HIAC (pink) within a 10.5-dpc embryo. (B) Relative distribution of multipotent precursors (orange), SAPs (purple), and HIACs (pink) in 8.5- to 12-dpc embryos. Whereas GATA-3⁺ SAPs appear below the aortic floor as soon as the first multipotent precursor is generated, HIACs are present only at the AGM stage, when the number of precursors generated reaches a peak. (C) Model for emergence and migration of embryonic hematopoietic precursors: HSCs (orange) generated in the SAP (1) migrate toward the aortic floor (2). HSC translocation, which contributes to HIAC development (3), is followed by the release of HIACs into the circulation.