Proinflammatory cytokine production by adherent donor blood cells stimulated by soluble LPS and phagocyted bacteria

Specific stimulation of receptors of the innate immune system by their purified ligands is commonly used in basic studies of inflammation and in the development of anti-inflammatory drugs. Based on location, receptors of the innate immunity can be classified into two groups: i) cell plasma membrane and on membranes of endosomes (Toll-like receptors (TLRs) and C-type lectin receptors), and recognizing the presence of pathogens in the extracellular space; ii) cytoplasmic sensors playing a special role in the recognition of intracellular pathogens (NOD-like receptors (NLRs), RIG-I-like receptors (RLRs), bacterial DNA sensor cGAS, and Aim2 (absent in melanoma 2). Many experimental models of inflammation use bacterial lipopolysaccharides (LPSs) or other purified microbial molecules to simulate the innate immune response to microbes. In the present study, the response of human blood leukocytes to stimulation with soluble, highly purified LPS from gram-negative bacteria was compared with that induced by formalin-fixed, corpuscular E. coli. The data obtained demonstrate that LPS and bacteria induce similar levels of TNF and IL-6 by plastic-adherent leukocytes, whereas neither LPS nor whole bacteria induce a measurable IFNγ production. The LPS- and bacteria-induced cytokine production, however, drastically differed in the sensitivity to a broad-spectrum TLR inhibitor, peptide 5R667. The LPS-stimulated human leukocyte cytokine production, as expected, was highly sensitive to inhibition by the peptide, whereas production stimulated by corpuscular bacteria was not. The TLR-blocking peptide did not affect the ability of blood leukocytes to phagocytose E. coli as shown by flow cytometry data obtained using FITC-stained fixed bacteria. Because peptide 5R667 blocks several TLRs, including TLR4, TLR5, and TLR9, the differential sensitivity of LPS- and bacteria-induced cytokine production to 5R667 suggests that the intracellular pathogen sensors, most likely NOD1 and/or NOD2, essentially contribute to the bacteria-induced cytokine induction. These results show that LPS and phagocyted bacteria induce cytokine production via different mechanisms and also suggest that the models with corpuscular bacteria for simulating bacterially induced inflammation complement the models that using soluble TLR ligands; therefore, both models should be applied to properly reflect anti-bacterial immune response

GFI1 proteins regulate stem cell formation in the AGM

In vertebrates, the first haematopoietic stem cells (HSCs) with multi-lineage and long-term repopulating potential arise in the AGM (aorta-gonad-mesonephros) region. These HSCs are generated from a rare and transient subset of endothelial cells, called haemogenic endothelium (HE), through an endothelial-to-haematopoietic transition (EHT). Here, we establish the absolute requirement of the transcriptional repressors GFI1 and GFI1B (growth factor independence 1 and 1B) in this unique trans-differentiation process. We first demonstrate that Gfi1 expression specifically defines the rare population of HE that generates emerging HSCs. We further establish that in the absence of GFI1 proteins, HSCs and haematopoietic progenitor cells are not produced in the AGM, revealing the critical requirement for GFI1 proteins in intra-embryonic EHT. Finally, we demonstrate that GFI1 proteins recruit the chromatin-modifying protein LSD1, a member of the CoREST repressive complex, to epigenetically silence the endothelial program in HE and allow the emergence of blood cells.We thank the staff at the Advanced Imaging, animal facility, Molecular Biology Core facilities and Flow Cytometry of CRUK Manchester Institute for technical support and Michael Lie-A-Ling and Elli Marinopoulou for initiating the DamID-PIP bioinformatics project. We thank members of the Stem Cell Biology group, the Stem Cell Haematopoiesis groups and Martin Gering for valuable advice and critical reading of the manuscript. Work in our laboratory is supported by the Leukaemia and Lymphoma Research Foundation (LLR), Cancer Research UK (CRUK) and the Biotechnology and Biological Sciences Research Council (BBSRC). SC is the recipient of an MRC senior fellowship (MR/J009202/1).This is the author accepted manuscript. The final version is available from NPG via http://dx.doi.org/10.1038/ncb327

Ethical and scientific aspects of human embryonic material research: The Great Britain regulations

We analyze the ethical and scientific issues of human embryonic specimen and its utilization for research purposes. Human embryonic specimens are the source of stem cells which are the foundation for all organs and tissue formation. The analysis of human embryonic specimens promises to obtain the essential knowledge about human development and reveal the causes of human development pathology. Ethical and scientific problems of human embryonic specimens donation and research are debated here. We review the impact that human embryonic specimens impose on scientific research, usage restrictions, risk assessment, research personnel and donor protection in view of the British, and other European publications, case reports, legislation, regulations and guidelines. This work is focused on the discussion of different aspects of donation and usage of human embryonic specimens obtained after the elective medical termination of pregnancy. We discuss suggestions for improvement of the ethical and scientific regulations for the work with human embryonic donor material in Russian Federation

Understanding Hematopoietic Stem Cell Development through Functional Correlation of Their Proliferative Status with the Intra-aortic Cluster Architecture

During development, hematopoietic stem cells (HSCs) emerge in the aorta-gonad-mesonephros (AGM) region through a process of multi-step maturation and expansion. While proliferation of adult HSCs is implicated in the balance between self-renewal and differentiation, very little is known about the proliferation status of nascent HSCs in the AGM region. Using Fucci reporter mice that enable in vivo visualization of cell-cycle status, we detect increased proliferation during pre-HSC expansion followed by a slowing down of cycling once cells start to acquire a definitive HSC state, similar to fetal liver HSCs. We observe time-specific changes in intra-aortic hematopoietic clusters corresponding to HSC maturation stages. The proliferative architecture of the clusters is maintained in an orderly anatomical manner with slowly cycling cells at the base and more actively proliferating cells at the more apical part of the cluster, which correlates with c-KIT expression levels, thus providing an anatomical basis for the role of SCF in HSC maturation

Murine hematopoietic stem cell activity is derived from pre-circulation embryos but not yolk sacs.

The embryonic site of definitive hematopoietic stem cell (dHSC) origination has been debated for decades. Although an intra-embryonic origin is well supported, the yolk sac (YS) contribution to adult hematopoiesis remains controversial. The same developmental origin makes it difficult to identify specific markers that discern between an intraembryonic versus YS-origin using a lineage trace approach. Additionally, the highly migratory nature of blood cells and the inability of pre-circulatory embryonic cells (i.e., 5-7 somite pairs (sp)) to robustly engraft in transplantation, even after culture, has precluded scientists from properly answering these questions. Here we report robust, multi-lineage and serially transplantable dHSC activity from cultured 2-7sp murine embryonic explants (Em-Ex). dHSC are undetectable in 2-7sp YS explants. Additionally, the engraftment from Em-Ex is confined to an emerging CD31+CD45+c-Kit+CD41- population. In sum, our work supports a model in which the embryo, not the YS, is the major source of lifelong definitive hematopoiesis

Intra-Aortic Clusters Undergo Endothelial to Hematopoietic Phenotypic Transition during Early Embryogenesis

Intra-aortic clusters (IACs) attach to floor of large arteries and are considered to have recently acquired hematopoietic stem cell (HSC)-potential in vertebrate early mid-gestation embryos. The formation and function of IACs is poorly understood. To address this issue, IACs were characterized by immunohistochemistry and flow cytometry in mouse embryos. Immunohistochemical analysis revealed that IACs simultaneously express the surface antigens CD31, CD34 and c-Kit. As embryos developed from 9.5 to 10.5 dpc, IACs up-regulate the hematopoietic markers CD41 and CD45 while down-regulating the endothelial surface antigen VE-cadherin/CD144, suggesting that IACs lose endothelial phenotype after 9.5 dpc. Analysis of the hematopoietic potential of IACs revealed a significant change in macrophage CFC activity from 9.5 to 10.5 dpc. To further characterize IACs, we isolated IACs based on CD45 expression. Correspondingly, the expression of hematopoietic transcription factors in the CD45(neg) fraction of IACs was significantly up-regulated. These results suggest that the transition from endothelial to hematopoietic phenotype of IACs occurs after 9.5 dpc

Inductive interactions mediated by interplay of asymmetric signalling underlie development of adult hematopoietic stem cells

During embryonic development, adult haematopoietic stem cells (HSCs) emerge preferentially in the ventral domain of the aorta in the aorta-gonad-mesonephros (AGM) region. Several signalling pathways such as Notch, Wnt, Shh and RA are implicated in this process, yet how these interact to regulate the emergence of HSCs has not previously been described in mammals. Using a combination of ex vivo and in vivo approaches, we report here that stage-specific reciprocal dorso-ventral inductive interactions and lateral input from the urogenital ridges are required to drive HSC development in the aorta. Our study strongly suggests that these inductive interactions in the AGM region are mediated by the interplay between spatially polarized signalling pathways. Specifically, Shh produced in the dorsal region of the AGM, stem cell factor in the ventral and lateral regions, and BMP inhibitory signals in the ventral tissue are integral parts of the regulatory system involved in the development of HSCs

Directed differentiation of embryonic stem cells using a bead-based combinatorial screening method

We have developed a rapid, bead-based combinatorial screening method to determine optimal combinations of variables that direct stem cell differentiation to produce known or novel cell types having pre-determined characteristics. Here we describe three experiments comprising stepwise exposure of mouse or human embryonic cells to 10,000 combinations of serum-free differentiation media, through which we discovered multiple novel, efficient and robust protocols to generate a number of specific hematopoietic and neural lineages. We further demonstrate that the technology can be used to optimize existing protocols in order to substitute costly growth factors with bioactive small molecules and/or increase cell yield, and to identify in vitro conditions for the production of rare developmental intermediates such as an embryonic lymphoid progenitor cell that has not previously been reported