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ABSTRACTrange of clinical pathologies, including idiopathic recurrent miscarriage, 19 fetal hemoglobin expression in Brazilian patients with sickle cell anemia, 20 and pre-eclampsia. 21Splenic macrophages are central to whole body iron recycling and return the iron from cleared RBC to the BM for use in erythropoiesis. 16,22 Hmox1 plays a critical role in this iron recycling and regulates the ability of splenic macrophages to tolerate the toxic heme released during RBC clearance. 16Hmox1 is expressed in splenic macrophages and is up-regulated in other cell types in response to heme and oxidative stress. 23 Splenic macrophages are significantly decreased in mice lacking Hmox1, 16 resulting in iron redistribution from the spleen and hepatic Küpffer cells to hepatocytes and proximal tubular cells of the kidney. 16 Inappropriate handling of heme and tissue deposition of iron in Hmox1 -/-mice and HMOX1-deficient patients results in increased oxidative stress and vascular cell injury. 16,17 Hematopoiesis and stress erythropoiesis in Hmox1 +/-mice has been investigated recently. BM cells from Hmox1 +/-mice are less capable of reconstituting lethally irradiated recipient animals. 24 Moreover, mice with a hematopoietic system reconstituted from Hmox1 +/-donor animals demonstrate decreased stress erythropoiesis in response to anemia. 25 Here, we investigated erythropoiesis in the complete absence of the Hmox1 gene and protein expression and without exerting exogenous stress in young, 8-to 14-week old mice. We found significant alterations in the BM, circulating and splenic erythroid populations in Hmox1 -/-mice. In the BM, the number of EBI macrophages was decreased and the expression of adhesion molecules was altered in erythroblast and macrophage populations, manifested as the inability of Hmox1-deficient BM to form EBI. Hmox1-deficient RBC also showed profound changes in redox biology and lifespan. Splenic erythropoiesis was almost completely absent and splenic macrophages involved in RBC removal were severely depleted. Our findings document defects in RBC production and lifespan resulting from the loss of this crucial heme-catabolizing enzyme. MethodsFull details of the materials and methods are provided in the Online Supplementary Methods. Mice Hmox1+/-breeders on a BALB/c background were obtained from Dr MP Soares (Instituto de Gulbenkian de Ciencia, Portugal) from a colony generated originally by Dr SF Yet. 26 Flow cytometry and cytological analysisBlood, BM, spleen and liver single cell suspensions were treated with FcR-block (MACS Miltenyi Biotech) then with appropriate antibodies for 1 h at 4°C. A list of antibodies is presented in Online Supplementary Table S1. The presence of a nucleus or surface membrane phosphatidylserine was assessed as described in the Online Supplementary Methods. Resuspended samples were analyzed using FACSCalibur (Becton Dickinson) and FlowJo software. Iron staining was performed on methanol-fixed cytospun BM samples using the Iron Stain Kit (Sigma Aldrich) according to the manufacturer...

Red blood cells, or erythrocytes, make up approximately a quarter of all cells in the human body with over 2 billion new erythrocytes made each day in a healthy adult human. This massive cellular production system is coupled with a set of cell biological processes unique to mammals, in particular, the elimination of all organelles, and the expulsion and destruction of the condensed erythroid nucleus. Erythrocytes from birds, reptiles, amphibians and fish possess nuclei, mitochondria and other organelles: erythrocytes from mammals lack all of these intracellular components. This review will focus on the dynamic changes that take place in developing erythroid cells that are interacting with specialized macrophages in multicellular clusters termed erythroblastic islands. Proerythroblasts enter the erythroblastic niche as large cells with active nuclei, mitochondria producing heme and energy, and attach to the central macrophage via a range of adhesion molecules. Proerythroblasts then mature into erythroblasts and, following enucleation, in reticulocytes. When reticulocytes exit the erythroblastic island, they are smaller cells, without nuclei and with few mitochondria, possess some polyribosomes and have a profoundly different surface molecule phenotype. Here, we will review, step-by-step, the biophysical mechanisms that regulate the remarkable process of erythropoiesis with a particular focus on the events taking place in the erythroblastic island niche. This is presented from the biological perspective to offer insight into the elements of red blood cell development in the erythroblastic island niche which could be further explored with biophysical modelling systems.

The detection of externalized phosphatidylserine (PS) on the cell surface is commonly used to distinguish between living, apoptotic and necrotic cells. The tools of choice for many researchers to study apoptosis are Annexin V-fluorophore conjugates. However, the use of this 35 kDa protein is associated with several drawbacks such as temperature sensitivity, Ca 2+ dependence, and slow binding kinetics. Here, we describe a fluorogenic probe for cell surface PS, P-IID, which operates by an intramolecular indicator displacement (IID) mechanism. An intramolecularly bound coumarin indicator is released in the presence of cell surface PS leading to a fluorescence 'turn-on' response. P-IID demonstrates superior performance when compared to Annexin V, for both fluorescence imaging and flow cytometry. In particular, P-IID binding to cell surfaces is not reliant on cells being at room temperature or the presence of calcium ions, does not require a wash step, and is significantly faster than that of Annexin V. This allows P-IID to be used in time-lapse imaging of apoptosis using confocal laser scanning microscopy and demonstrates the utility of the IID mechanism in live cells for the first time.

Approximately one-quarter of all cells in the adult human body are blood cells. The haematopoietic system is therefore massive in scale and requires exquisite regulation to be maintained under homeostatic conditions. It must also be able to respond when needed, such as during infection or following blood loss, to produce more blood cells. Supporting cells serve to maintain haematopoietic stem and progenitor cells during homeostatic and pathological conditions. This coalition of supportive cell types, organised in specific tissues, is termed the haematopoietic niche. Haematopoietic stem and progenitor cells are generated in a number of distinct locations during mammalian embryogenesis. These stem and progenitor cells migrate to a variety of anatomical locations through the conceptus until finally homing to the bone marrow shortly before birth. Under stress, extramedullary haematopoiesis can take place in regions that are typically lacking in blood-producing activity. Our aim in this review is to examine blood production throughout the embryo and adult, under normal and pathological conditions, to identify commonalities and distinctions between each niche. A clearer understanding of the mechanism underlying each haematopoietic niche can be applied to improving ex vivo cultures of haematopoietic stem cells and potentially lead to new directions for transplantation medicine.