Loss of tRNA-modifying enzyme Elp3 activates a p53-dependent antitumor checkpoint in hematopoiesis.

The hematopoietic system is highly sensitive to perturbations in the translational machinery, of which an emerging level of regulation lies in the epitranscriptomic modification of transfer RNAs (tRNAs). Here, we interrogate the role of tRNA anticodon modifications in hematopoiesis by using mouse models of conditional inactivation of Elp3, the catalytic subunit of Elongator that modifies wobble uridine in specific tRNAs. Loss of Elp3 causes bone marrow failure by inducing death in committing progenitors and compromises the grafting activity of hematopoietic stem cells. Mechanistically, Elp3 deficiency activates a p53-dependent checkpoint in what resembles a misguided amino acid deprivation response that is accompanied by Atf4 overactivation and increased protein synthesis. While deletion of p53 rescues hematopoiesis, loss of Elp3 prompts the development of p53-mutated leukemia/lymphoma, and inactivation of p53 and Elongator cooperatively promotes tumorigenesis. Specific tRNA-modifying enzymes thus condition differentiation and antitumor fate decisions in hematopoietic stem cells and progenitors.

Detection of Hypoxic Regions in the Bone Microenvironment.

Oxygen serves as a critical environmental factor essential for maintaining the physiological state of a tissue. Hypoxia, or low oxygen, triggers a cascade of events which allows for cells to adapt to low oxygen tensions and to facilitate oxygen delivery required to maintain tissue homeostasis. In the bone microenvironment (BME), vascular heterogeneity, poor perfusion rates of blood vessels, and high metabolic activity of hematopoietic cells result in the generation of a unique hypoxic landscape. Importantly, in this region, hypoxia and its downstream effectors are associated with establishing stem cell niches and regulating the differentiation of committed progenitors. Given the functional importance of the hypoxic bone niche, visualizing regions of hypoxia may provide valuable insights into the mechanisms that regulate tissue homeostasis. Here, we describe the utilization of the nitroimidazole derivative, pimonidazole, to detect hypoxic regions within the BME.

Post-transcriptional control of cellular differentiation by the RNA exosome complex.

Given the complexity of intracellular RNA ensembles and vast phenotypic remodeling intrinsic to cellular differentiation, it is instructive to consider the role of RNA regulatory machinery in controlling differentiation. Dynamic post-transcriptional regulation of protein-coding and non-coding transcripts is vital for establishing and maintaining proteomes that enable or oppose differentiation. By contrast to extensively studied transcriptional mechanisms governing differentiation, many questions remain unanswered regarding the involvement of post-transcriptional mechanisms. Through its catalytic activity to selectively process or degrade RNAs, the RNA exosome complex dictates the levels of RNAs comprising multiple RNA classes, thereby regulating chromatin structure, gene expression and differentiation. Although the RNA exosome would be expected to control diverse biological processes, studies to elucidate its biological functions and how it integrates into, or functions in parallel with, cell type-specific transcriptional mechanisms are in their infancy. Mechanistic analyses have demonstrated that the RNA exosome confers expression of a differentiation regulatory receptor tyrosine kinase, downregulates the telomerase RNA component TERC, confers genomic stability and promotes DNA repair, which have considerable physiological and pathological implications. In this review, we address how a broadly operational RNA regulatory complex interfaces with cell type-specific machinery to control cellular differentiation.

Daily Ethanol Drinking Followed by an Abstinence Period Impairs Bone Marrow Niche and Mitochondrial Function of Hematopoietic Stem/Progenitor Cells in Rhesus Macaques.

BACKGROUND: Unhealthy consumption of alcohol is a major public health crisis with strong associations between immunological dysfunctions, high vulnerability to infectious disease, anemia, and an increase in the risk of hematological malignancies. However, there is a lack of studies addressing alcohol-induced changes in bone marrow (BM) and hematopoiesis as fundamental aspects of immune system function. METHODS: To address the effect of chronic alcohol consumption on hematopoietic stem and progenitor cells (HSPCs) and the BM niche, we used an established rhesus macaque model of voluntary alcohol drinking. A cohort of young adult male rhesus macaques underwent a standard ethanol self-administration protocol that allowed a choice of drinking alcohol or water 22 hours/day with periods of forced abstinence that elevated subsequent intakes when alcohol availability resumed. Following the last month of forced abstinence, the monkeys were euthanized. HSPCs and bone samples were collected and analyzed in functional assays and by confocal microscopy. RESULTS: HSPCs from alcohol animals exhibited reduced ability to form granulocyte-monocyte and erythroid colonies in vitro. HSPCs also displayed a decrease in mitochondrial oxygen consumption linked to ATP production and basal respiratory capacity. Chronic alcohol use led to vascular remodeling of the BM niche, a reduction in the number of primitive HSPCs, and a shift in localization of HSPCs from an adipose to a perivascular niche. CONCLUSIONS: Our study demonstrates, for the first time, that chronic voluntary alcohol drinking in rhesus macaque monkeys leads to the long-term impairment of HSPC function, a reduction in mitochondrial respiratory activity, and alterations in the BM microenvironment. Further studies are needed to determine whether these changes in hematopoiesis are persistent or adaptive during the abstinent period and whether an initial imprinting to alcohol primes BM to become more vulnerable to future exposure to alcohol.

Identification of miRNAs from stem cell derived microparticles in umbilical cord blood.

Umbilical cord blood CD34+ (UCB-CD34+) stem cells are clinically used in hematopoietic cell transplantation. However, there are limitations in the use of umbilical cord blood transplants because of the small number of cells and delayed engraftment. To gain a better understanding of functional components of UCB, we have detected and characterized CD34+ microparticles (CD34+MPs) from cord blood units. We collected cord blood units and assessed the numbers of CD34+MPs before and after red blood cell and plasma depletion by SEPAX processing using flow cytometry analysis. In parallel we identified MPs by electron microscopy. CD34+MPs and cells were isolated by MACs sorting. MicroRNAs (miR-106, miR-221, miR-517, miR-519, and miR-221) exhibited a characteristic microRNA profile that was further validated in isolated CD34+MPs. We found that in cord blood, there are CD34+MPs that carry microRNAs.

Static spatial growth restriction micropatterning of endothelial colony forming cells influences their morphology and gene expression.

BACKGROUND: Endothelialization of small diameter synthetic vascular grafts is a potential solution to the thrombosis and intimal hyperplasia that plague current devices. Endothelial colony forming cells, which are blood-derived and similar to mature endothelial cells, are a potential cell source. Anisotropic spatial growth restriction micropatterning has been previously shown to affect the morphology and function of mature endothelial cells in a manner similar to unidirectional fluid shear stress. To date, endothelial colony forming cells have not been successfully micropatterned. This study addresses the hypothesis that micropatterning of endothelial colony forming cells will induce morphological elongation, cytoskeletal alignment, and changes in immunogenic and thrombogenic-related gene expression. METHODS: Spatially growth restrictive test surfaces with 25 µm-wide lanes alternating between collagen-I and a blocking polymer were created using microfluidics. Case-matched endothelial colony forming cells and control mature carotid endothelial cells were statically cultured on either micropatterned or non-patterned surfaces. Cell elongation was quantified using shape index. Using confocal microscopy, cytoskeletal alignment was visualized and density and apoptotic rate were determined. Gene expression was measured using quantitative PCR to measure KLF-2, eNOS, VCAM-1, and vWF. RESULTS: Endothelial colony forming cells were successfully micropatterned for up to 50 hours. Micropatterned cells displayed elongation and actin alignment. Micropatterning increased the packing densities of both cell types, but did not affect apoptotic rate, which was lower in endothelial colony forming cells. KLF-2 gene expression was increased in micropatterned relative to non-patterned endothelial colony forming cells after 50 hours. No significant differences were seen in the other genes tested. CONCLUSIONS: Endothelial colony forming cells can be durably micropatterned using spatial growth restriction. Micropatterning has a significant effect on the gross and subcellular morphologies of both cell types. Further study is required to fully understand the effect of micropatterning on endothelial colony forming cell gene expression.

One cell one niche: hematopoietic microenvironments constructed by bone marrow stromal cells with fibroblastic and histiocytic features.

Hematopoietic microenvironments have been extensively studied, especially focusing on regulation of hematopoietic stem cells (HSCs) in HSC niche following progress of molecular biology in resent years. Based on prior morphological achievements from 1970s, the characteristics of cellular compartments and bone marrow stromal cells (BMSCs) were studied ultrastructurally in human and mice bone marrow in the present study. The samples, human bone marrow granules, were collected from bone marrow aspirations (BMAs) of 20 patients with hematocytopenia and isolated BMSCs were found undesignedly in nucleated cells of BMAs of the patients. Femoral bone marrow samples were collected from 6-week-old three sacrificed mice. Detailed images illustrated maturing hematopoietic cells harbored individually in honeycomb-like microenvironment constituted by BMSCs that shared of fibroblastic and histiocytic characteristics in hematopoietic microenvironments of human and mice bone marrow.

Measurement of Mitochondrial Mass and Membrane Potential in Hematopoietic Stem Cells and T-cells by Flow Cytometry.

A fine balance of quiescence, self-renewal, and differentiation is key to preserve the hematopoietic stem cell (HSC) pool and maintain lifelong production of all mature blood cells. In recent years cellular metabolism has emerged as a crucial regulator of HSC function and fate. We have previously demonstrated that modulation of mitochondrial metabolism influences HSC fate. Specifically, by chemically uncoupling the electron transport chain we were able to maintain HSC function in culture conditions that normally induce rapid differentiation. However, limiting HSC numbers often precludes the use of standard assays to measure HSC metabolism and therefore predict their function. Here, we report a simple flow cytometry assay that allows reliable measurement of mitochondrial membrane potential and mitochondrial mass in scarce cells such as HSCs. We discuss the isolation of HSCs from mouse bone marrow and measurement of mitochondrial mass and membrane potential post ex vivo culture. As an example, we show the modulation of these parameters in HSCs via treatment with a metabolic modulator. Moreover, we extend the application of this methodology on human peripheral blood-derived T cells and human tumor infiltrating lymphocytes (TILs), showing dramatic differences in their mitochondrial profiles, possibly reflecting different T cell functionality. We believe this assay can be employed in screenings to identify modulators of mitochondrial metabolism in various cell types in different contexts.

Thrombopoietin Metabolically Primes Hematopoietic Stem Cells to Megakaryocyte-Lineage Differentiation.

During acute myelosuppression or thrombocytopenia, bone marrow (BM) hematopoietic cells respond rapidly to replenish peripheral blood platelets. While the cytokine thrombopoietin (Thpo) both regulates platelet production and maintains HSC potential, whether Thpo controls megakaryocyte (Mk)-lineage differentiation of HSCs is unclear. Here, we show that Thpo rapidly upregulates mitochondrial activity in HSCs, an activity accompanied by differentiation to an Mk lineage. Moreover, in unperturbed hematopoiesis, HSCs with high mitochondrial activity exhibit Mk-lineage differentiation in vitro and myeloid lineage-biased reconstitution in vivo. Furthermore, Thpo skewed HSCs to express the tetraspanin CD9, a pattern correlated with mitochondrial activity. Mitochondria-active HSCs are resistant to apoptosis and oxidative stress upon Thpo stimulation. Thpo-regulated mitochondrial activity associated with mitochondrial translocation of STAT3 phosphorylated at serine 727. Overall, we report an important role for Thpo in regulating rapid Mk-lineage commitment. Thpo-dependent changes in mitochondrial metabolism prime HSCs to undergo direct differentiation to an Mk lineage.

Current practices for viability testing of cryopreserved cord blood products: an international survey by the cellular therapy team of the Biomedical Excellence for Safer Transfusion (BEST) Collaborative.

BACKGROUND: Viability testing is a common practice in laboratories. The goal of this study was to ascertain current laboratory practices internationally for performing viability testing for cryopreserved cord blood (CB) products and glean information about how to standardize the method to improve interlaboratory reproducibility. STUDY DESIGN AND METHODS: A survey to evaluate current laboratory practices for viability testing was designed and distributed internationally. The question topics included sampling and testing methods, responses to unexpected results, and the rating of the reliability of the CB quality tests, together with expectations for standardization. RESULTS: There were 32 respondents to the survey, of whom 28 responded to the more detailed questionnaire about viability methods. Overall, responses indicated that various stains were used among the laboratories, and when multiple sites used the same viability stain the methods differed. The majority of the respondents were in favor of standardizing the viability testing methods. A wide variety of preferences were communicated about how to standardize the method, but a majority did advocate the use of 7-aminoactinomycin D (7-AAD) with flow cytometry. CONCLUSIONS: The survey results revealed a variety of tests and inconsistent interlaboratory practices for performing the viability assay. Flow cytometry with a 7-AAD dye was suggested as a first step toward standardization.

Quantitative spatial analysis of haematopoiesis-regulating stromal cells in the bone marrow microenvironment by 3D microscopy.

Sinusoidal endothelial cells and mesenchymal CXCL12-abundant reticular cells are principal bone marrow stromal components, which critically modulate haematopoiesis at various levels, including haematopoietic stem cell maintenance. These stromal subsets are thought to be scarce and function via highly specific interactions in anatomically confined niches. Yet, knowledge on their abundance, global distribution and spatial associations remains limited. Using three-dimensional quantitative microscopy we show that sinusoidal endothelial and mesenchymal reticular subsets are remarkably more abundant than estimated by conventional flow cytometry. Moreover, both cell types assemble in topologically complex networks, associate to extracellular matrix and pervade marrow tissues. Through spatial statistical methods we challenge previous models and demonstrate that even in the absence of major specific interaction forces, virtually all tissue-resident cells are invariably in physical contact with, or close proximity to, mesenchymal reticular and sinusoidal endothelial cells. We further show that basic structural features of these stromal components are preserved during ageing.

The telopode- and filopode-projecting heterogeneous stromal cells of the human sclera niche.

Telocytes (TCs) are stromal cells defined by the presence of long and slender prolongations (telopodes). They are a biologically and functionally heterogeneous population that has not been previously investigated in the sclera. The purpose of this study is to investigate the presence and characteristics of scleral telocytes through a combined immunohistochemical and transmission electron microscopy (TEM) study using samples from ten adult patients. Stromal cells with a TC-like morphology expressed CD34, CD45, CD105, vimentin and occasionally CD68 but were negative for collagen III, CD31, CD133, and CD146. Conjunctival epithelial cells expressed CD45, CD105, CD146, and vimentin. These phenotypes support a scleral niche with immune TCs and haematopoietic stem cells (HSCs). In TEM, we often found spindle-shaped stromal cells projecting telopodes or filopodes, with extremely long nuclei extended even within those prolongations. We separated these cells into a light subtype, which contained a complete set of organelles, and a dark subtype, consisting of undifferentiated stem/progenitor cells. The light cells contained dense vesicles, Weibel-Palade bodies, and rounded α-granule-like structures. These storage areas for the von Willebrand factor (vWF) are known to express selectins that are critically involved in HSC homing and could also indicate endothelial progenitors. The dark cells were scarcely myoid, populated the episcleral perivascular niches and the scleral stroma, and were equipped with lipid storage areas such as lamellar bodies and lipid droplets (LDs). Previously, unreported intranuclear LDs were found in these cells, which is characteristic of an HSC population. It appears that the human scleral stroma is a niche harbouring TC-like cells with immune and HSC phenotypes, and the mere presence or characteristics of telopodes are not enough to differentiate them.

Bone marrow-on-a-chip: Long-term culture of human haematopoietic stem cells in a three-dimensional microfluidic environment.

Multipotent haematopoietic stem and progenitor cells (HSPCs) are the source for all blood cell types. The bone marrow stem cell niche in which the HSPCs are maintained is known to be vital for their maintenance. Unfortunately, to date, no in vitro model exists that accurately mimics the aspects of the bone marrow niche and simultaneously allows the long-term culture of HSPCs. In this study, a novel three-dimensional coculture model is presented, based on a hydroxyapatite coated zirconium oxide scaffold, comprising of human mesenchymal stromal cells (MSCs) and cord blood derived HSPCs, enabling successful HSPC culture for a time span of 28 days within the microfluidic multiorgan chip. The HSPCs were found to stay in their primitive state (CD34+ CD38- ) and capable of granulocyte, erythrocyte, macrophage, megakaryocyte colony formation. Furthermore, a microenvironment was formed bearing molecular and structural similarity to the in vivo bone marrow niche containing extracellular matrix and signalling molecules known to play an important role in HSPC homeostasis. Here, a novel human in vitro bone marrow model is presented for the first time, capable of long-term culture of primitive HSPCs in a microfluidic environment.

PDK1 plays a vital role on hematopoietic stem cell function.

3-Phosphoinositide-dependent protein kinase 1 (PDK1) is a pivotal regulator in the phosphoinositide 3-kinase (PI3K)-Akt signaling pathway that have been shown to play key roles in the functional development of B and T cells via activation of AGC protein kinases during hematopoiesis. However, the role of PDK1 in HSCs has not been fully defined. Here we specifically deleted the PDK1 gene in the hematopoietic system and found that PDK1-deficient HSCs exhibited impaired function and defective lineage commitment abilities. Lack of PDK1 caused HSCs to be less quiescent and to produce a higher number of phenotypic HSCs and fewer progenitors. PDK1-deficient HSCs were also unable to reconstitute the hematopoietic system. Notably, HSC function was more dependent on PDK1 than on mTORC2, which indicates that PDK1 plays a dominant role in the Akt-mediated regulation of HSC function. PDK1-deficient HSCs also exhibited reduced ROS levels, and treatment of PDK1-deficient HSCs with L-butathioninesulfoximine in vitro elevated the low ROS level and promoted colony formation. Therefore, PDK1 appears to contribute to HSC function partially via regulating ROS levels.

Bone marrow hematons: An access point to the human hematopoietic niche.

To understand the complex interactions between hematopoietic stem cells and the bone marrow niche, a human experimental model is needed. Our hypothesis is that hematons are an appropriate ex vivo model of human bone marrow. We analyzed the hierarchical hematopoietic cell content and the tissue organization of single hematons from healthy donors. Most (>90%) hematons contained precursors of all cell lineages, myeloid progenitors, and LTC-ICs without preferential commitment. Approximately, half of the hematons could generate significant levels of lympho-myeloid hematopoiesis after transplantation in an NSG mouse model, despite the low absolute numbers of transplanted CD34+ cells. Mesenchymal STRO-1+ and/or CD271+ cells formed a critical network that preserved hematon cohesion, and STRO-1+ cells colocalized with most hematopoietic CD34+ cells (68%). We observed an influence of age and gender. These structures represent a particularly attractive model for studying the homeostasis of the bone marrow niche and pathological changes that occur during diseases.

Quantitative analyses of the 3D nuclear landscape recorded with super-resolved fluorescence microscopy.

Recent advancements of super-resolved fluorescence microscopy have revolutionized microscopic studies of cells, including the exceedingly complex structural organization of cell nuclei in space and time. In this paper we describe and discuss tools for (semi-) automated, quantitative 3D analyses of the spatial nuclear organization. These tools allow the quantitative assessment of highly resolved different chromatin compaction levels in individual cell nuclei, which reflect functionally different regions or sub-compartments of the 3D nuclear landscape, and measurements of absolute distances between sites of different chromatin compaction. In addition, these tools allow 3D mapping of specific DNA/RNA sequences and nuclear proteins relative to the 3D chromatin compaction maps and comparisons of multiple cell nuclei. The tools are available in the free and open source R packages nucim and bioimagetools. We discuss the use of masks for the segmentation of nuclei and the use of DNA stains, such as DAPI, as a proxy for local differences in chromatin compaction. We further discuss the limitations of 3D maps of the nuclear landscape as well as problems of the biological interpretation of such data.

Characterization of putative haematopoietic cells from bovine yolk sac.

The yolk sac is an extra-embryonic membrane that plays an important role in early embryonic survival. It is the production site for blood cells during embryonic mammalian development and is a likely source of stem cells. The aim of this study was to identify and characterize the putative haematopoietic cells from the yolk sac of bovine embryos at different stages of gestation. The yolk sac regresses according to gestational age and embryos are characterized into groups (I-V) according to the crown-rump measurement. Groups I-III survived in culture longer and exhibited the formation of cell clusters, whereas groups IV and V could not be maintained in culture for an extended period of time. Flow-cytometry analysis revealed that groups I-III had similar characteristics, including high expression levels of the haematopoietic markers CD34, CD90 and CD117. In groups IV and V, decreases were observed in the expression levels of CD117 and CD34. Cells were found to be capable of survival post-cryopreservation and exhibited varying abilities to form colonies in a methylcellulose matrix, depending on gestational age. Cytological analysis revealed the presence of blood cells (lymphocytes and monocytes). Quantitative PCR analysis demonstrated the presence of the haematopoietic progenitor genes GATA3 and LMO2, but not RUNX1. Thus, we have successfully isolated and characterized haematopoietic cells from the bovine embryo yolk sac at varying gestational ages. This study is crucial for the understanding of the development of the haematopoietic system and the embryonic function of this organ. Copyright © 2015 John Wiley & Sons, Ltd.

Nitrogen-containing bisphosphonate induces a newly discovered hematopoietic structure in the omentum of an anemic mouse model by stimulating G-CSF production.

We previously reported that the injection of nitrogen-containing bisphosphonate (NBP) induced the site of erythropoiesis to shift from the bone marrow (BM) to the spleen. Our previous study established a severely anemic mouse model that was treated with a combination of NBP with phenylhydrazine (PHZ), which induced newly discovered hematopoietic organs in the omentum. No reports have shown that new hematopoietic organs form under any condition. We characterized the structures and factors related to the formation of these new organs. Splenectomized mice were treated with NBP to inhibit erythropoiesis in the BM and then injected with PHZ to induce hemolytic anemia. The mice showed severe anemia and wine-colored structures appeared in the omentum. Some hematopoietic cells, including megakaryocytes, and well-developed sinuses were observed in these structures. Numerous TER119-positive erythroblasts were located with cells positive for PCNA, a cell proliferation marker. C-kit-positive cells were detected and mRNAs related to hematopoiesis were expressed in these structures. Moreover, TER119-positive erythroblasts emerged and formed clusters and hematopoiesis-related factors were detected in the omentum of mice treated with NBP and PHZ. The levels of G-CSF in the serum and hematopoietic progenitor cells (HPCs) in the peripheral blood were increased upon treatment with both NBP and PHZ. These results suggest that the induced hematopoietic structures act as the sites of erythropoiesis and that NBP-induced G-CSF production causes HPC mobilization, homing and colonization in the omentum because they constitutively express some factors, including SDF-1; thus, the newly discovered hematopoietic structure in this study might be formed.

Tunneling nanotubes mediate the transfer of stem cell marker CD133 between hematopoietic progenitor cells.

Deciphering all mechanisms of intercellular communication used by hematopoietic progenitors is important, not only for basic stem cell research, but also in view of their therapeutic relevance. Here, we investigated whether these cells can produce the thin F-actin-based plasma membrane protrusions referred to as tunneling nanotubes (TNTs), which are known to bridge cells over long distances without contact with the substratum and transfer cargo molecules along them in various biological processes. We found that human primary CD34+ hematopoietic progenitors and leukemic KG1a cells develop such structures upon culture on primary mesenchymal stromal cells or specific extracellular-matrix-based substrata. Time-lapse video microscopy revealed that cell dislodgement is the primary mechanism responsible for TNT biogenesis. Surprisingly, we found that, among various cluster of differentiation (CD) markers, only the stem cell antigen CD133 is transferred between cells. It is selectively and directionally transported along the surface of TNTs in small clusters, such as cytoplasmic phospho-myosin light chain 2, suggesting that the latter actin motor protein might be implicated in this process. Our data provide new insights into the biology of hematopoietic progenitors that can contribute to our understanding of all facets of intercellular communication in the bone marrow microenvironment under healthy or cancerous conditions.

Histiocytic differentiation in acute monocytic leukemia.

Myeloid histocytes of dendritic cells (DCs), Langerhans cells (LCs), and macrophages in varied tissues, as leukemic blasts in acute monoblastic and monocytic leukemia (AML-M5a and M5b), are derived from monocyte progenitors in bone marrow. Based on DC induction from hematopoietic stem cells, myeloid progenitors, and monocytes, and occasional expressions of histocyte-related antigens (HRAs) in M5, we presume some M5 cases share histiocytic phenotypes originally. To clarify the conception, 93 M5 cases were tested with antibodies for HRAs, CD1a, CD163, S100, fascin, and langerin by immunostaining, and their morphologic characteristics were studied by light and transmission electron microscopy. The study revealed that 23 M5 cases were positive for two or more kinds of HRAs and shared a serial of histocytic immunophenotype and morphologic features, which were closely associated with M5b subtype and expression of CD14 in M5.