FOXF1 inhibits hematopoietic lineage commitment during early mesoderm specification.

The molecular mechanisms orchestrating early mesoderm specification are still poorly understood. In particular, how alternate cell fate decisions are regulated in nascent mesoderm remains mostly unknown. In the present study, we investigated both in vitro in differentiating embryonic stem cells, and in vivo in gastrulating embryos, the lineage specification of early mesodermal precursors expressing or not the Forkhead transcription factor FOXF1. Our data revealed that FOXF1-expressing mesoderm is derived from FLK1(+) progenitors and that in vitro this transcription factor is expressed in smooth muscle and transiently in endothelial lineages, but not in hematopoietic cells. In gastrulating embryos, FOXF1 marks most extra-embryonic mesoderm derivatives including the chorion, the allantois, the amnion and a subset of endothelial cells. Similarly to the in vitro situation, FOXF1 expression is excluded from the blood islands and blood cells. Further analysis revealed an inverse correlation between hematopoietic potential and FOXF1 expression in vivo with increased commitment toward primitive erythropoiesis in Foxf1-deficient embryos, whereas FOXF1-enforced expression in vitro was shown to repress hematopoiesis. Altogether, our data establish that during gastrulation, FOXF1 marks all posterior primitive streak extra-embryonic mesoderm derivatives with the remarkable exception of the blood lineage. Our study further suggests that this transcription factor is implicated in actively restraining the specification of mesodermal progenitors to hematopoiesis.

VE-cadherin expression allows identification of a new class of hematopoietic stem cells within human embryonic liver.

Edification of the human hematopoietic system during development is characterized by the production of waves of hematopoietic cells separated in time, formed in distinct embryonic sites (ie, yolk sac, truncal arteries including the aorta, and placenta). The embryonic liver is a major hematopoietic organ wherein hematopoietic stem cells (HSCs) expand, and the future, adult-type, hematopoietic cell hierarchy becomes established. We report herein the identification of a new, transient, and rare cell population in the human embryonic liver, which coexpresses VE-cadherin, an endothelial marker, CD45, a pan-hematopoietic marker, and CD34, a common endothelial and hematopoietic marker. This population displays an outstanding self-renewal, proliferation, and differentiation potential, as detected by in vitro and in vivo hematopoietic assays compared with its VE-cadherin negative counterpart. Based on VE-cadherin expression, our data demonstrate the existence of 2 phenotypically and functionally separable populations of multipotent HSCs in the human embryo, the VE-cadherin(+) one being more primitive than the VE-cadherin(-) one, and shed a new light on the hierarchical organization of the embryonic liver HSC compartment.

Monoclonal antibody 1.6.1 against human MPL receptor allows HSC enrichment of CB and BM CD34(+)CD38(-) populations.

Thrombopoietin (TPO) and its receptor Mpl (CD110) play a crucial role in the regulation of hematopoietic stem cells (HSCs). Functional study of Mpl-expressing HSCs has, however, been hampered by the lack of efficient monoclonal antibodies, explaining the very few data available on Mpl(+) HSCs during human embryonic development and after birth. Investigating the main monoclonal antibodies used so far to sort CD110(+) cells from cord blood (CB) and adult bone marrow (BM), we found that only the recent monoclonal antibody 1.6.1 engineered by Immunex Corporation was specific. Using in vitro functional assays, we found that this antibody can be used to sort a CD34(+)CD38(-)CD110(+) population enriched in hematopoietic progenitor stem cells, both in CB and in adult BM. In vivo injection into NSG mice further indicated that the CB CD34(+)CD38(-)CD110(+) population is highly enriched in HSCs compared with both CD34(+)CD38(-)CD110(-) and CD34(+)CD38(-) populations. Together our results validate MAb1.6.1 as an important tool, which has so far been lacking, in the HSC field.

In vivo repopulating activity emerges at the onset of hematopoietic specification during embryonic stem cell differentiation.

The generation of in vivo repopulating hematopoietic cells from in vitro differentiating embryonic stem cells has remained a long-standing challenge. To date, hematopoietic engraftment has mostly been achieved through the enforced expression of ectopic transcription factors. Here, we describe serum-free culture conditions that allow the generation of in vivo repopulating hematopoietic cells in the absence of ectopically expressed factors. We show that repopulating activity arises immediately upon the commitment of mesodermal precursors to the blood program, within the first wave of hematopoietic specification. We establish that the formation of these progenitors is extremely transient and exquisitely sensitive to the cytokine milieu. Our findings define the precise differentiating stage at which hematopoietic repopulating activity first appears in vitro, and suggest that during embryonic stem cell differentiation, all hematopoietic programs are unraveled simultaneously from the mesoderm in the absence of cues that restrict the coordinated emergence of each lineage as is normally observed during embryogenesis.

Long-lasting inhibitory effects of fetal liver mesenchymal stem cells on T-lymphocyte proliferation.

Human bone marrow mesenchymal stem cells (BM-MSC) are multipotent progenitor cells that have transient immunomodulatory properties on Natural Killer (NK) cells, Dendritic Cells (DC), and T cells. This study compared the use of MSC isolated from bone marrow and fetal liver (FL-MSC) to determine which displayed the most efficient immunosuppressive effects on T cell activation. Although both types of MSC exhibit similar phenotype profile, FL-MSC displays a much more extended in vitro life-span and immunomodulatory properties. When co-cultured with CD3/CD28-stimulated T cells, both BM-MSC and FL-MSC affected T cell proliferation by inhibiting their entry into the cell cycle, by inducing the down-regulation of phospho-retinoblastoma (pRb), cyclins A and D1, as well as up-regulating p27(kip1) expression. The T cell inhibition by MSC was not due to the soluble HLA-G5 isoform, but to the surface expression of HLA-G1, as shown by the need of cell-cell contact and by the use of neutralizing anti-HLA-G antibodies. To note, in a HLA-G-mediated fashion, MSC facilitated the expansion of a CD4(low)/CD8(low) T subset that had decreased secretion of IFN-γ, and an induced secretion of the immunomodulatory cytokine IL-10. Because of their longer lasting in vitro immunosuppressive properties, mainly mediated by HLA-G, and their more efficient induction of IL-10 production and T cell apoptosis, fetal liver MSC could be considered a new tool for MSC therapy to prevent allograft rejection.

Mpl receptor defect leads to earlier appearance of hematopoietic cells/hematopoietic stem cells in the Aorta-Gonad-Mesonephros region, with increased apoptosis.

In a previous study, we underlined the functional role of the TPO receptor, Mpl, in the establishment of definitive mouse hematopoiesis, by demonstrating that the lack of Mpl led to a delayed production of definitive hematopoietic cells in the aorta-gonad-mesonephros (AGM) region, and resulted in the production of hematopoietic stem cells (HSCs) with an impaired activity at E11.5. In order to more accurately estimate the role of Mpl during generation of HSCs in the aorta, we performed an analysis of these AGMs at the time of the first HSC emergence (E10.5). Our results indicated that while Mpl-/- AGMs were found to contain more hematopoietic cells (HC) than C57Bl6 AGMs at E10.5, a defect in the expansion process of the HC/HSCs was detected in explant cultures of these AGMs, likely due to an increased apoptosis of these cells. To determine the molecular mechanisms by which invalidation of Mpl receptor affects the temporal distribution and expansion of HC/HSCs in the AGM, a study of the transcription level of of Mpl target genes was conducted. Expression of Runx1, a master transcription factor for the formation of hematopoietic progenitor (HP) cells and HSCs from the vasculature, as well as expression of Meis1 and HoxB4, known to play a role in self-renewal and expansion of HSCs, were found to be down regulated in E10.5 Mpl-/- AGMs. Our data indicate that Mpl is an active player during the first steps of definitive hematopoiesis establishment through direct regulation of the expression of transcription factors or genes important for the self-renewal, proliferation and apoptosis of HSCs.

Dual role of Mpl receptor during the establishment of definitive hematopoiesis.

Cytokine signaling pathways are important in promoting hematopoietic stem cell (HSC) self-renewal, proliferation and differentiation. Mpl receptor and its ligand, TPO, have been shown to play an essential role in the early steps of adult hematopoiesis. We previously demonstrated that the cytoplasmic domain of Mpl promotes hematopoietic commitment of embryonic stem cells in vitro, and postulated that Mpl could be important in the establishment of definitive hematopoiesis. To answer this question, we investigated the temporal expression of Mpl during mouse development by in situ hybridization. We found Mpl expression in the HSCs clusters emerging in the AGM region, and in the fetal liver (FL) as early as E10.5. Using Mpl(-/-) mice, the functional relevance of Mpl expression was tested by comparing the hematopoietic progenitor (HP) content, long-term hematopoietic reconstitution (LTR) abilities and HSC content of control and Mpl(-/-) embryos at different times of development. In the AGM, we observed delayed production of HSCs endowed with normal LTR but presenting a self-renewal defect. During FL development, we detected a decrease in HP and HSC potential associated with a defect in amplification and self-renewal/survival of the lin(-) AA4.1(+) Sca1(+) population of HSCs. These results underline the dual role of Mpl in the generation and expansion of HSCs during establishment of definitive hematopoiesis.