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.

Thrombopoietin-increased DNA-PK-dependent DNA repair limits hematopoietic stem and progenitor cell mutagenesis in response to DNA damage.

DNA double-strand breaks (DSBs) represent a serious threat for hematopoietic stem cells (HSCs). How cytokines and environmental signals integrate the DNA damage response and contribute to HSC-intrinsic DNA repair processes remains unknown. Thrombopoietin (TPO) and its receptor, Mpl, are critical factors supporting HSC self-renewal and expansion. Here, we uncover an unknown function for TPO-Mpl in the regulation of DNA damage response. We show that DNA repair following γ-irradiation (γ-IR) or the action of topoisomerase-II inhibitors is defective in Mpl(-/-) and in wild-type mouse or human hematopoietic stem and progenitor cells treated in the absence of TPO. TPO stimulates DNA repair in vitro and in vivo by increasing DNA-PK-dependent nonhomologous end-joining efficiency. This ensures HSC chromosomal integrity and limits their long-term injury in response to IR. This shows that niche factors can modulate the HSC DSB repair machinery and opens new avenues for administration of TPO agonists for minimizing radiotherapy-induced HSC injury and mutagenesis.

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

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.

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.

Definitive human and mouse hematopoiesis originates from the embryonic endothelium: a new class of HSCs based on VE-cadherin expression.

Hematopoietic stem cells (HSCs) arise first in the third week of human ontogeny inside yolk sac developing blood vessels, and independently, from the wall of the embryonic aorta and vitelline arteries one week later. HSCs produced in the yolk sac and in the embryonic truncal arteries migrate to and transiently colonize the embryonic liver (EL), and thereafter the bone marrow (BM), their permanent site of residence. At the moment, the origin of human HSCs is still controversial; one of the main hypotheses being that they are generated by hemogenic endothelial cells (ECs). To prove definitively the endothelial origin of HSCs that arise within the human embryo, we previously purified ECs from either the yolk sac or the truncal arteries and reported that they were able to produce blood cells in vitro. We then found that some of the HSCs present in the human EL were co-expressing vascular endothelial (VE)-cadherin, an endothelial marker, CD45, a pan-hematopoietic marker, and CD34, a common endothelial and hematopoietic marker, and demonstrated that these HSCs bearing a dual hemato-endothelial phenotype were endowed with remarkably high self renewal and proliferative potentials. Furthermore, a transgenic mouse model based on the VE-cadherin cis-regulating elements that we engineered to trace the fate of the first VE-cadherin expressing cells allowed us to clearly demonstrate that a majority of adult BM HSCs derived from a VE-cadherin ancestor. Altogether our studies strongly suggest that at least a part of both the human and the murine hematopoietic systems arise from an endothelium-like ancestor.

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.

Glycosaminoglycan mimetics-induced mobilization of hematopoietic progenitors and stem cells into mouse peripheral blood: structure/function insights.

OBJECTIVE: Glycosaminoglycans (GAG) are major components of bone marrow extracellular matrix because they have the property to interact with cells and growth factors in hematopoietic niches. In this study, we investigated the effect of two different chemically defined GAG mimetics on mobilization of hematopoietic stem and progenitor cells (HSPCs) in mice peripheral blood. MATERIALS AND METHODS: Mobilization was achieved by intraperitoneal injection of GAG mimetics. Mobilized cells were characterized phenotypically by reverse transcription polymerase chain reaction and fluorescence-activated cell sorting analysis and functionally by colony-forming cell, cobblestone area-forming cell and long-term culture-initiating cell assays in vitro. Radioprotection assays were performed to confirm the functionality of primitive hematopoietic cells in vivo. Involvement of stromal-derived factor-1 (SDF-1) and matrix metalloproteinase-9 (MMP-9) were investigated. RESULTS: GAG mimetics treatment induces hyperleukocytosis and mobilization of HSPC. They synergize with the effects of granulocyte colony-stimulating factor or AMD3100 on hematopoietic progenitors mobilization. Reconstitution of lethally irradiated recipient mice with peripheral blood mononuclear cells from GAG mimetic-treated donor mice improves engraftment and survival. BiAcore studies indicate that the mimetics interact directly with SDF-1. In addition, GAG mimetics-induced mobilization is associated with increased levels of pro- and active MMP-9 from bone marrow cells and increased level of SDF-1 in peripheral blood. Finally, mobilization is partially inhibited by co-injection with anti-SDF-1 antibody. CONCLUSION: This study demonstrates that GAG mimetics induce efficient mobilization of HSPCs, associated with an activation of pro-MMP-9 and a modification in the SDF-1 concentration gradient between bone marrow and peripheral blood. We suggest that structural features of GAGs can modify the nature of mobilized cells.

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.