Pigs share many physiological, biochemical, and anatomical similarities with humans and have emerged as valuable large animal models for biomedical research. Considering the advantages in immune system resemblance, suitable size, and longevity for clinical practical and monitoring purpose, SCID pigs bearing dysfunctional RAG could serve as important experimental tools for regenerative medicine, allograft and xenograft transplantation, and reconstitution experiments related to the immune system. In this study, we report the generation and phenotypic characterization of RAG1 and RAG2 knockout pigs using transcription activator-like effector nucleases. Porcine fetal fibroblasts were genetically engineered using transcription activator-like effector nucleases and then used to provide donor nuclei for somatic cell nuclear transfer. We obtained 27 live cloned piglets; among these piglets, 9 were targeted with biallelic mutations in RAG1, 3 were targeted with biallelic mutations in RAG2, and 10 were targeted with a monoallelic mutation in RAG2. Piglets with biallelic mutations in either RAG1 or RAG2 exhibited hypoplasia of immune organs, failed to perform V(D)J rearrangement, and lost mature B and T cells. These immunodeficient RAG1/2 knockout pigs are promising tools for biomedical and translational research.
The transcription factor c-Myb was originally identified as a transforming oncoprotein encoded by two avian leukaemia viruses. Subsequently, through the generation of mouse models that affect its expression, c-Myb has been shown to be a key regulator of hematopoiesis, including having critical roles in hematopoietic stem cells (HSC). The precise function of c-Myb in HSCs though remains unclear. We have generated a novel c-myb allele in mice that allows direct observation of c-Myb protein levels in single cells. Using this reporter line we demonstrate that subtypes of HSCs can be isolated based upon their respective c-Myb protein expression levels. HSCs expressing low levels of c-Myb protein (c-Myb(low) HSC) appear to represent the most immature, dormant HSCs and they are a predominant component of HSCs that retain bromodeoxyuridine (BrdU) labelling. Hematopoietic stress, induced by 5-fluorouracil ablation, revealed that in this circumstance c-Myb-expressing cells become critical for multilineage repopulation. The discrimination of HSC subpopulations based on c-Myb protein levels is not reflected in the levels of c-myb mRNA, there being no more than a 1.3-fold difference comparing c-Myb(low) and c-Myb(high) HSCs. This illustrates how essential it is to include protein studies when aiming to understand the regulatory networks that control stem cell behaviour. Stem Cells 2014.
Reactivation of the pluripotency network during somatic cell reprogramming by exogenous transcription factors involves chromatin remodeling and the recruitment of RNA polymerase II (Pol II) to target loci. Here, we report that Pol II is engaged at pluripotency promoters in reprogramming but remains paused and inefficiently released. We also show that bromodomain-containing protein 4 (BRD4) stimulates productive transcriptional elongation of pluripotency genes by dissociating the pause release factor P-TEFb from an inactive complex containing HEXIM1. Consequently, BRD4 overexpression enhances reprogramming efficiency and HEXIM1 suppresses it, whereas Brd4 and Hexim1 knockdown do the opposite. We further demonstrate that the reprogramming factor KLF4 helps recruit P-TEFb to pluripotency promoters. Our work thus provides a mechanism for explaining the reactivation of pluripotency genes in reprogramming and unveils an unanticipated role for KLF4 in transcriptional pause release.
Zinc-finger nucleases and transcription activator-like effector nucleases are novel gene-editing platforms contributing to redefine the boundaries of modern biological research. They are composed of a non-specific cleavage domain and a tailor made DNA-binding module, which enables a broad range of genetic modifications by inducing efficient DNA double-strand breaks at desired loci. Among other remarkable uses, these nucleases have been employed to produce gene knockouts in mid-size and large animals, such as rabbits and pigs, respectively. This approach is cost effective, relatively quick, and can produce invaluable models for human disease studies, biotechnology or agricultural purposes. Here we describe a protocol for the efficient generation of knockout rabbits using transcription activator-like effector nucleases, and a perspective of the field.
A novel method is presented for accurately reconstructing a spatially resolved map of diffuse light flux on a surface using images of the surface and a model of the imaging system. This is achieved by applying a model-based reconstruction algorithm with an existing forward model of light propagation through free space that accounts for the effects of perspective, focus, and imaging geometry. It is shown that flux can be mapped reliably and quantitatively accurately with very low error, <3% with modest signal-to-noise ratio. Simulation shows that the method is generalizable to the case in which mirrors are used in the system and therefore multiple views can be combined in reconstruction. Validation experiments show that physical diffuse phantom surface fluxes can also be reconstructed accurately with variability <3% for a range of object positions, variable states of focus, and different orientations. The method provides a new way of making quantitatively accurate noncontact measurements of the amount of light leaving a diffusive medium, such as a small animal containing fluorescent or bioluminescent markers, that is independent of the imaging system configuration and surface position.
Haematopoietic stem cells (HSCs) are the founding cells of the adult haematopoietic system, born during ontogeny from a specialized subset of endothelium, the haemogenic endothelium (HE) via an endothelial-to-haematopoietic transition (EHT). Although recently imaged in real time, the underlying mechanism of EHT is still poorly understood. We have generated a Runx1 +23 enhancer-reporter transgenic mouse (23GFP) for the prospective isolation of HE throughout embryonic development. Here we perform functional analysis of over 1,800 and transcriptional analysis of 268 single 23GFP(+) HE cells to explore the onset of EHT at the single-cell level. We show that initiation of the haematopoietic programme occurs in cells still embedded in the endothelial layer, and is accompanied by a previously unrecognized early loss of endothelial potential before HSCs emerge. Our data therefore provide important insights on the timeline of early haematopoietic commitment.
Mesenchymal stem cells (MSC) have immuno-modulatory properties, but their effects on endothelial cells (EC) and recruitment of leukocytes are unknown. We cocultured human bone marrow-derived MSC with EC, and found that MSC could down-regulate adhesion of flowing neutrophils or lymphocytes, and their subsequent transendothelial migration. This applied for EC treated with tumour necrosis factor-? (TNF), interleukin-1? (IL-1) or TNF and interferon-? combined. Supernatant from cocultures also inhibited endothelial responses. This supernatant had much higher levels of IL-6 than supernatant from cultures of the individual cells, which also lacked inhibitory functions. Addition of neutralising antibody against IL-6 removed the bioactivity of the supernatant and also the immunomodulatory effects of coculture. Studies using siRNA showed that IL-6 came mainly from the MSC in coculture, and reduction in production in MSC alone was sufficient to impair the protective effects of coculture. Interestingly, siRNA knock-down of IL-6-receptor expression in MSC as well as EC inhibited anti-inflammatory effects. This was explained when we detected soluble IL-6R-receptor in supernatants and showed that receptor removal reduced the potency of supernatant. Neutralisation of transforming growth factor-? indicated that activation of this factor in coculture contributed to Il-6 production. Thus, crosstalk between MSC and EC caused up regulation of production of IL-6 by MSC which in turn down regulated the response of EC to inflammatory cytokines, an effect potentiated by MSC release of soluble IL-6R. These studies establish a novel mechanism by which MSC might have protective effects against inflammatory pathology and cardiovascular disease. Stem Cells 2013.
Although haematopoietic stem cells (HSCs) migrate to injured gut, therapeutic success clinically remains poor. This has been partially attributed to limited local HSC recruitment following systemic injection. Identifying site specific adhesive mechanisms underpinning HSC-endothelial interactions may provide important information on how to enhance their recruitment and thus potentially improve therapeutic efficacy. This study determined (i) the integrins and inflammatory cyto/chemokines governing HSC adhesion to injured gut and muscle (ii) whether pre-treating HSCs with these cyto/chemokines enhanced their adhesion and (iii) whether the degree of HSC adhesion influenced their ability to modulate leukocyte recruitment.
The aorta-gonad-mesonephros region plays an important role in hematopoietic stem cell (HSC) development during mouse embryogenesis. The vascular endothelial cadherin? CD45? (VE-cad?CD45?) population contains the major type of immature pre-HSCs capable of developing into long-term repopulating definitive HSCs. In this study, we developed a new coaggregation culture system, which supports maturation of a novel population of CD45-negative (VE-cad?CD45?CD41?) pre-HSCs into definitive HSCs. The appearance of these pre-HSCs precedes development of the VE-cad?CD45? pre-HSCs (termed here type I and type II pre-HSCs, respectively), thus establishing a hierarchical directionality in the developing HSC lineage. By labeling the luminal surface of the dorsal aorta, we show that both type I and type II pre-HSCs are distributed broadly within the endothelial and subendothelial aortic layers, in contrast to mature definitive HSCs which localize to the aortic endothelial layer. In agreement with expression of CD41 in pre-HSCs, in vivo CD41-Cre-mediated genetic tagging occurs in embryonic pre-HSCs and persists in all lymphomyeloid lineages of the adult animal.
A common feature of early embryo cells from the inner cell mass (ICM) and of ESCs is an absolute dependence on an atypical cell cycle in which the G1 phase is shortened to preserve their self-renewing and pluripotent nature. The transcription factor B-Myb has been attributed a role in proliferation, in particular during the G2/M phases of the cell cycle. Intriguingly, B-Myb levels in ICM/ESCs are greater than 100 times compared with those in normal proliferating cells, suggesting a particularly important function for this transcription factor in pluripotent stem cells. B-Myb is essential for embryo development beyond the preimplantation stage, but its role in ICM/ESCs remains unclear. Using a combination of mouse genetics, single DNA fiber analyses and high-resolution three-dimensional (3D) imaging, we demonstrate that B-Myb has no influence on the expression of pluripotency factors, but instead B-Myb ablation leads to stalling of replication forks and superactivation of replication factories that result in disorganization of the replication program and an increase in double-strand breaks. These effects are partly due to aberrant transcriptional regulation of cell cycle proliferation factors, namely c-Myc and FoxM1, which dictate normal S phase progression. We conclude that B-Myb acts crucially during the S phase in ESCs by facilitating proper progression of replication, thereby protecting the cells from genomic damage. Our findings have particular relevance in the light of the potential therapeutic application of ESCs and the need to maintain their genomic integrity.
c-Myb is a transcription factor with functions in many hematopoietic lineages. c-Myb-deficient mice display reduced numbers of B cells; however, it is unknown what role c-Myb plays in B lymphopoiesis because no critical target genes have been identified in the B-cell lineage. We demonstrate that conditional deletion of c-Myb in B-cell progenitors completely abolishes B-cell development. c-Myb is required for lymphoid progenitors to respond to the cytokines interleukin-7 and thymic stromal lymphopoietin; in the absence of sufficient c-Myb activity, mice display a B lymphopenia that closely resembles that observed in interleukin-7 receptor alpha-deficient animals. Analysis of the multipotent progenitor compartment indicates that c-Myb is also required for up-regulation of multiple lymphoid-associated genes, including Il7r, and for the subsequent development of the common lymphoid progenitor population. These data show that c-Myb plays a critical role in the regulatory pathways governing lymphoid specification and early B-cell differentiation.
The ductus arteriosus (DA) is a fetal shunt vessel between the pulmonary artery and the aorta that closes promptly after birth. Failure of postnatal DA closure is a major cause of morbidity and mortality particularly in preterm neonates. The events leading to DA closure are incompletely understood. Here we show that platelets have an essential role in DA closure. Using intravital microscopy of neonatal mice, we observed that platelets are recruited to the luminal aspect of the DA during closure. DA closure is impaired in neonates with malfunctioning platelet adhesion or aggregation or with defective platelet biogenesis. Defective DA closure resulted in a left-to-right shunt with increased pulmonary perfusion, pulmonary vascular remodeling and right ventricular hypertrophy. Our findings indicate that platelets are crucial for DA closure by promoting thrombotic sealing of the constricted DA and by supporting luminal remodeling. A retrospective clinical study revealed that thrombocytopenia is an independent predictor for failure of DA closure in preterm human newborns, indicating that platelets are likely to contribute to DA closure in humans.
Integrin alpha IIb beta 3 is expressed in mast cells as well as in megakaryocytes/platelets. A recent study has shown that surface expression levels of integrin alpha V beta 3 are elevated in integrin alpha IIb-deficient bone marrow-derived mast cells (BMMCs) as compared with wild-type (WT) counterparts, but the underlying mechanism remains obscure. Here we demonstrate by transducing integrin alpha IIb into integrin alpha IIb-deficient BMMCs that surface expression levels of integrin alpha V beta 3 are inversely related to those of integrin alpha IIb beta 3. Thus, competitive association of integrin beta 3 with integrin alpha IIb or integrin alpha V determines surface expression levels of integrin alpha IIb beta 3 or alpha V beta 3 in mast cells. We compared WT and integrin alpha IIb-deficient BMMCs as well as integrin alpha IIb-deficient BMMCs transduced with integrin alpha IIb(WT) or non-functional alpha IIb(D163A) mutant and found that enhancement of proliferation, degranulation, cytokine production, and migration of BMMCs through interaction with fibrinogen (FB) depended on integrin alpha IIb beta 3. In addition, elevated surface expression of integrin alpha V beta 3 failed to compensate for loss of FB-associated functions in integrin alpha IIb-deficient BMMCs while enhancing adhesion to vitronectin or von Willebrand factor. Importantly, integrin alpha IIb deficiency strongly suppressed chronic inflammation with the remarkable increase of mast cells induced by continuous intraperitoneal administration of FB, although it did not affect acute allergic responses or mast cell numbers in tissues in steady states. Interestingly, soluble FB promoted cytokine production of BMMCs in response to Staphylococcus aureus with FB-binding capacity, through integrin alpha IIb beta 3-dependent recognition of this pathogen. Collectively, integrin alpha IIb beta 3 in mast cells plays an important part in FB-associated, chronic inflammation and innate immune responses.
Murine haematopoietic stem cells (HSCs) are contained in the Kit+Sca1+Lin(-) (KSL) population of bone marrow and are able to repopulate lethally irradiated mice. Myeloproliferative disorders (MPDs) are thought to be clonogenic diseases arising at the level of the HSC. Here, we show that mice expressing low levels of the transcription factor c-Myb, as the result of genetic knockdown, develop a transplantable myeloproliferative phenotype that closely resembles the human disease essential thrombocythaemia (ET). Unlike wild-type cells, the KSL population in c-myb knockdown bone marrow cannot repopulate irradiated mice and does not transfer the disease. Instead, cells positive for Kit and expressing low to medium levels of CD11b acquire self-renewing stem cell properties and are responsible for the perpetuation of the myeloproliferative phenotype.
A disintegrin and metalloprotease 10 (ADAM10) is a ubiquitous transmembrane metalloprotease that cleaves the extracellular regions from over 40 different transmembrane target proteins, including Notch and amyloid precursor protein. ADAM10 is essential for embryonic development and is also important in inflammation, cancer, and Alzheimer disease. However, ADAM10 regulation remains poorly understood. ADAM10 is compartmentalized into membrane microdomains formed by tetraspanins, which are a superfamily of 33 transmembrane proteins in humans that regulate clustering and trafficking of certain other transmembrane "partner" proteins. This is achieved by specific tetraspanin-partner interactions, but it is not clear which tetraspanins specifically interact with ADAM10. The aims of this study were to identify which tetraspanins interact with ADAM10 and how they regulate this metalloprotease. Co-immunoprecipitation identified specific ADAM10 interactions with Tspan5, Tspan10, Tspan14, Tspan15, Tspan17, and Tspan33/Penumbra. These are members of the largely unstudied TspanC8 subgroup of tetraspanins, all six of which promoted ADAM10 maturation. Different cell types express distinct repertoires of TspanC8 tetraspanins. Human umbilical vein endothelial cells express relatively high levels of Tspan14, the knockdown of which reduced ADAM10 surface expression and activity. Mouse erythrocytes express predominantly Tspan33, and ADAM10 expression was substantially reduced in the absence of this tetraspanin. In contrast, ADAM10 expression was normal on Tspan33-deficient mouse platelets in which Tspan14 is the major TspanC8 tetraspanin. These results define TspanC8 tetraspanins as essential regulators of ADAM10 maturation and trafficking to the cell surface. This finding has therapeutic implications because focusing on specific TspanC8-ADAM10 complexes may allow cell type- and/or substrate-specific ADAM10 targeting.
Product of the Itga2b gene, CD41 contributes to hematopoietic stem cell (HSC) and megakaryocyte/platelet functions. CD41 expression marks the onset of definitive hematopoiesis in the embryo where it participates in regulating the numbers of multipotential progenitors. Key to platelet aggregation, CD41 expression also characterises their precursor, the megakaryocyte, and is specifically up regulated during megakaryopoiesis. Though phenotypically unique, megakaryocytes and HSC share numerous features, including key transcription factors, which could indicate common sub-regulatory networks. In these respects, Itga2b can serve as a paradigm to study features of both developmental-stage and HSC- versus megakaryocyte-specific regulations. By comparing different cellular contexts, we highlight a mechanism by which internal promoters participate in Itga2b regulation. A developmental process connects epigenetic regulation and promoter switching leading to CD41 expression in HSC. Interestingly, a similar process can be observed at the Mpl locus, which codes for another receptor that defines both HSC and megakaryocyte identities. Our study shows that Itga2b expression is controlled by lineage-specific networks and associates with H4K8ac in megakaryocyte or H3K27me3 in the multipotential hematopoietic cell line HPC7. Correlating with the decrease in H3K27me3 at the Itga2b Iocus, we find that following commitment to megakaryocyte differentiation, the H3K27 demethylase Jmjd3 up-regulation influences both Itga2b and Mpl expression.
Hematopoietic stem cells (HSCs) migrate to injury sites and aid in tissue repair. However, clinical success is poor and is partially due to limited HSC recruitment. We hypothesized that HSC pretreatment with H2O2 would enhance their recruitment to injured gut. As HSCs are rare cells, the number of primary cells obtained from donors is often inadequate for functional experiments. To circumvent this, in this study we utilized a functionally relevant cell line, HPC-7. Anesthetized mice were subjected to intestinal ischemia-reperfusion (IR) injury, and HPC-7 recruitment was examined intravitally. Adhesion to endothelial cells (ECs), injured gut sections, and ICAM-1/VCAM-1 protein were also quantitated in vitro. H2O2 pretreatment significantly enhanced HPC-7 recruitment to injured gut in vivo. A concomitant reduction in pulmonary adhesion was also observed. Enhanced adhesion was also observed in all in vitro models. Increased clustering of ?4 and ?2 integrins, F-actin polymerization, and filopodia formation were observed in pretreated HPC-7s. Importantly, H2O2 did not reduce HPC-7 viability or proliferative ability. HPC-7 recruitment to injured gut can be modulated by H2O2 pretreatment. This may be through increasing the affinity or avidity of surface integrins that mediate HPC-7 homing to injured sites or through stimulating the migratory apparatus. Strategies that enhance hematopoietic stem/progenitor cell recruitment may ultimately affect their therapeutic efficacy.
Macrophages and dendritic cells (DCs) are key components of cellular immunity and are thought to originate and renew from hematopoietic stem cells (HSCs). However, some macrophages develop in the embryo before the appearance of definitive HSCs. We thus reinvestigated macrophage development. We found that the transcription factor Myb was required for development of HSCs and all CD11b(high) monocytes and macrophages, but was dispensable for yolk sac (YS) macrophages and for the development of YS-derived F4/80(bright) macrophages in several tissues, such as liver Kupffer cells, epidermal Langerhans cells, and microglia--cell populations that all can persist in adult mice independently of HSCs. These results define a lineage of tissue macrophages that derive from the YS and are genetically distinct from HSC progeny.
Human bone marrow mesenchymal stem cells (hMSCs) have shown benefit in clinical trials of patients with liver disease. Efficient delivery of cells to target organs is critical to improving their effectiveness. This requires an understanding of the mechanisms governing cellular engraftment into the liver. Binding of hMSCs to normal/injured liver tissue, purified extracellular matrices, and human hepatic sinusoidal endothelial cells (HSECs) were quantified in static and flow conditions. To define the mechanisms underpinning hMSC interactions, neutralizing adhesion molecule antibodies were used. Fluorescently labelled hMSCs were infused intraportally into CCl(4) -injured mice with and without neutralizing antibodies. hMSCs expressed high levels of CD29/?1-integrin and CD44. Using liver tissue binding assays, hMSC adhesion was greatest in diseased human liver versus normal liver (32.2 cells/field versus 20.5 cells/field [P = 0.048]). Neutralizing antibodies against CD29 and CD44 reduced hMSC binding to diseased liver by 34% and 35%, respectively (P = 0.05). hMSCs rolled at 528 ?m/second on HSECs in flow assays. This rolling was abolished by CD29 blockade on hMSCs and vascular cell adhesion molecule-1 (VCAM-1) blockade on HSECs. Firm adhesion to HSECs was reduced by CD29 (55% [P = 0.002]) and CD44 (51% [P = 0.04]) blockade. Neutralizing antibodies to CD29 and CD44 reduced hepatic engraftment of hMSCs in murine liver from 4.45 cells/field to 2.88 cells/field (P = 0.025) and 2.35 cells/field (P = 0.03), respectively. hMSCs expressed modest levels of chemokine receptors including CCR4, CCR5, and CXCR3, but these made little contribution to hMSC adhesion in this setting.
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