Scale bars represent 80 m. bone marrow HSC and human peripheral blood HSC-myeloid progenitors cultured in the presence of limited cytokine concentrations. Megakaryocytes obtained in V+P? cocultures were polyploid, positive for CD41/CD42c, and efficiently produced proplatelets. Megakaryocyte production appeared to be mediated by an expansion of the progenitor compartment through HSCCstromal cell contact. In conclusion, the fetal liver contains a unique cellular microenvironment that could represent a platform for the discovery of regulators of megakaryopoiesis. Visual Abstract Open in a separate window Introduction The proliferation and differentiation of hematopoietic stem cells (HSCs) are regulated by a Mouse monoclonal to CD10.COCL reacts with CD10, 100 kDa common acute lymphoblastic leukemia antigen (CALLA), which is expressed on lymphoid precursors, germinal center B cells, and peripheral blood granulocytes. CD10 is a regulator of B cell growth and proliferation. CD10 is used in conjunction with other reagents in the phenotyping of leukemia microenvironment combining Carbamazepine cellular and extracellular components, such as extracellular matrices, growth factors, and other biomolecules, which collectively exert their influence on HSC maintenance and differentiation. A particular microenvironment that regulates the self-renewal and the maintenance of Carbamazepine HSCs is also referred as the stem cell niche, a concept first proposed by R. Schofield.1 The cellular elements constituting the niche were first identified among the fibroblastic cells that form colonies in the appropriate conditions (colony-forming unit fibroblast [CFU-F]).2 CFU-F initiating cells and their progeny are also referred to as mesenchymal stem cells or stromal precursor cells. While significant progress in understanding the mechanisms involved in the maintenance of a self-renewing HSC has been achieved, very few studies have focused on the identification of the microenvironment regulating the commitment toward a given lineage, particularly the megakaryocytic lineage. This question is of great interest when considering our limited ability to reproduce in culture the megakaryopoiesis and thrombopoiesis efficiencies of the native environment. Reports considering the role of bone marrow stromal cells are conflicting with respect to their capacity to support megakaryopoiesis. Some studies suggest that contact with stromal cell precursors negatively controls megakaryocytic differentiation of the human hematopoietic cell line K5623,4 or human CD34 progenitors,5,6 whereas other studies suggest that stromal cells support or enhance megakaryopoiesis.7-9 This apparent contradiction may reside in differences in Carbamazepine experimental design and in the complexity of the processes involved in the generation of megakaryocytes (MKs) from HSCs. Indeed, MKs are generated from HSCs through multiple steps of committed MK progenitors, including a bipotent megakaryocytic erythroid progenitor (MEP), leading to the production of a unipotent MK precursor, which will then mature into large polyploid MKs that will extend proplatelets in the circulation. How, where and which stromal precursor cells intervene in this complex but well-orchestrated process is still subject to questions. Stromal cells produce a number of hematopoietic cytokines and other soluble factors regulating megakaryopoiesis.10 The major cytokine regulating megakaryopoiesis, thrombopoietin (TPO), stimulates the production of MKs, but not the final maturation: proplatelet production.11 This highlights the fact that the factors or cellular elements controlling the different steps of megakaryopoiesis are bound to be different from the commitment of HSCs toward the MK lineage and during the maturation of MK progenitors and precursors. Similarly, it is likely that different stages of MK expansion and maturation are regulated by distinct cellular microenvironments, and different hematopoietic tissues can be considered to explore this question. Megakaryopoiesis mainly occurs in the Carbamazepine bone marrow in adults but is also observed during embryogenesis. In the embryo, megakaryopoiesis proceeds following colonization of the fetal liver by HSCs originating in the aorta-gonad-mesonephros and possibly also by MK progenitors already present in the yolk sac.12 Large mature MKs are observed in the fetal liver from around 13 days of development in the mouse13 (Manuela Tavian, INSERM UMR S949, oral communication, 16 November 2015). The Carbamazepine fetal liver therefore represents an attractive tissue to study the microenvironment supporting the different stages of megakaryopoiesis. In this study, we characterized and isolated different stromal cell populations from mouse fetal liver with different functional properties. We found that a particular population with a hepatocyte progenitor signature supported efficient expansion of MK-committed progenitors able to produce fully mature MKs. Materials and methods Isolation of fetal liver stromal cells Pregnant females from timed breeding protocol were killed using CO2 inhalation followed by cervical dislocation. Fetuses were harvested, and the fetal liver was dissected under a binocular microscope. Fetal liver cell suspensions were obtained after digestion with 3 mg/mL collagenase I (Worthington Biochemical, Freehold, NJ) for 10 min at 37C, dilution with PBS-2% newborn serum, and filtration through a 70 m cell strainer (BD Biosciences, San Jose, CA). Fetal liver hematopoietic cells were depleted after labeling with biotinylated TER119 and anti-mouse CD45 antibodies using.
