Stem cell-derived porcine macrophages as a new platform for studying host-pathogen interactions.

BACKGROUND: Infectious diseases of farmed and wild animals pose a recurrent threat to food security and human health. The macrophage, a key component of the innate immune system, is the first line of defence against many infectious agents and plays a major role in shaping the adaptive immune response. However, this phagocyte is a target and host for many pathogens. Understanding the molecular basis of interactions between macrophages and pathogens is therefore crucial for the development of effective strategies to combat important infectious diseases. RESULTS: We explored how porcine pluripotent stem cells (PSCs) can provide a limitless in vitro supply of genetically and experimentally tractable macrophages. Porcine PSC-derived macrophages (PSCdMs) exhibited molecular and functional characteristics of ex vivo primary macrophages and were productively infected by pig pathogens, including porcine reproductive and respiratory syndrome virus (PRRSV) and African swine fever virus (ASFV), two of the most economically important and devastating viruses in pig farming. Moreover, porcine PSCdMs were readily amenable to genetic modification by CRISPR/Cas9 gene editing applied either in parental stem cells or directly in the macrophages by lentiviral vector transduction. CONCLUSIONS: We show that porcine PSCdMs exhibit key macrophage characteristics, including infection by a range of commercially relevant pig pathogens. In addition, genetic engineering of PSCs and PSCdMs affords new opportunities for functional analysis of macrophage biology in an important livestock species. PSCs and differentiated derivatives should therefore represent a useful and ethical experimental platform to investigate the genetic and molecular basis of host-pathogen interactions in pigs, and also have wider applications in livestock.

LIF-dependent survival of embryonic stem cells is regulated by a novel palmitoylated Gab1 signalling protein.

The cytokine leukaemia inhibitory factor (LIF) promotes self-renewal of mouse embryonic stem cells (ESCs) through activation of the transcription factor Stat3. However, the contribution of other ancillary pathways stimulated by LIF in ESCs, such as the MAPK and PI3K pathways, is less well understood. We show here that naive-type mouse ESCs express high levels of a novel effector of the MAPK and PI3K pathways. This effector is an isoform of the Gab1 (Grb2-associated binder protein 1) adaptor protein that lacks the N-terminal pleckstrin homology (PH) membrane-binding domain. Although not essential for rapid unrestricted growth of ESCs under optimal conditions, the novel Gab1 variant (Gab1ß) is required for LIF-mediated cell survival under conditions of limited nutrient availability. This enhanced survival is absolutely dependent upon a latent palmitoylation site that targets Gab1ß directly to ESC membranes. These results show that constitutive association of Gab1 with membranes through a novel mechanism promotes LIF-dependent survival of murine ESCs in nutrient-poor conditions.

Hypoxanthine phosphoribosyltransferase (HPRT)-deficiency is associated with impaired fertility in the female rat.

The purine hypoxanthine plays important role in regulating oocyte maturation and early embryonic development. The enzyme hypoxanthine phosphoribosyltransferase (HPRT) recycles hypoxanthine to generate substrates for nucleotide synthesis and key metabolites, and here we show that HPRT deficiency in the rat disrupts early embryonic development and causes infertility in females.

Pleiotropic Impacts of Macrophage and Microglial Deficiency on Development in Rats with Targeted Mutation of the Csf1r Locus.

We have produced Csf1r-deficient rats by homologous recombination in embryonic stem cells. Consistent with the role of Csf1r in macrophage differentiation, there was a loss of peripheral blood monocytes, microglia in the brain, epidermal Langerhans cells, splenic marginal zone macrophages, bone-associated macrophages and osteoclasts, and peritoneal macrophages. Macrophages of splenic red pulp, liver, lung, and gut were less affected. The pleiotropic impacts of the loss of macrophages on development of multiple organ systems in rats were distinct from those reported in mice. Csf1r-/- rats survived well into adulthood with postnatal growth retardation, distinct skeletal and bone marrow abnormalities, infertility, and loss of visceral adipose tissue. Gene expression analysis in spleen revealed selective loss of transcripts associated with the marginal zone and, in brain regions, the loss of known and candidate novel microglia-associated transcripts. Despite the complete absence of microglia, there was little overt phenotype in brain, aside from reduced myelination and increased expression of dopamine receptor-associated transcripts in striatum. The results highlight the redundant and nonredundant functions of CSF1R signaling and of macrophages in development, organogenesis, and homeostasis.

From engineering to editing the rat genome.

Since its domestication over 100 years ago, the laboratory rat has been the preferred experimental animal in many areas of biomedical research (Lindsey and Baker The laboratory rat. Academic, New York, pp 1-52, 2006). Its physiology, size, genetics, reproductive cycle, cognitive and behavioural characteristics have made it a particularly useful animal model for studying many human disorders and diseases. Indeed, through selective breeding programmes numerous strains have been derived that are now the mainstay of research on hypertension, obesity and neurobiology (Okamoto and Aoki Jpn Circ J 27:282-293, 1963; Zucker and Zucker J Hered 52(6):275-278, 1961). Despite this wealth of genetic and phenotypic diversity, the ability to manipulate and interrogate the genetic basis of existing phenotypes in rat strains and the methodology to generate new rat models has lagged significantly behind the advances made with its close cousin, the laboratory mouse. However, recent technical developments in stem cell biology and genetic engineering have again brought the rat to the forefront of biomedical studies and enabled researchers to exploit the increasingly accessible wealth of genome sequence information. In this review, we will describe how a breakthrough in understanding the molecular basis of self-renewal of the pluripotent founder cells of the mammalian embryo, embryonic stem (ES) cells, enabled the derivation of rat ES cells and their application in transgenesis. We will also describe the remarkable progress that has been made in the development of gene editing enzymes that enable the generation of transgenic rats directly through targeted genetic modifications in the genomes of zygotes. The simplicity, efficiency and cost-effectiveness of the CRISPR/Cas gene editing system, in particular, mean that the ability to engineer the rat genome is no longer a limiting factor. The selection of suitable targets and gene modifications will now become a priority: a challenge where ES culture and gene editing technologies can play complementary roles in generating accurate bespoke rat models for studying biological processes and modelling human disease.

An interspecies analysis reveals a key role for unmethylated CpG dinucleotides in vertebrate Polycomb complex recruitment.

The role of DNA sequence in determining chromatin state is incompletely understood. We have previously demonstrated that large chromosomal segments from human cells recapitulate their native chromatin state in mouse cells, but the relative contribution of local sequences versus their genomic context remains unknown. In this study, we compare orthologous chromosomal regions for which the human locus establishes prominent sites of Polycomb complex recruitment in pluripotent stem cells, whereas the corresponding mouse locus does not. Using recombination-mediated cassette exchange at the mouse locus, we establish the primacy of local sequences in the encoding of chromatin state. We show that the signal for chromatin bivalency is redundantly encoded across a bivalent domain and that this reflects competition between Polycomb complex recruitment and transcriptional activation. Furthermore, our results suggest that a high density of unmethylated CpG dinucleotides is sufficient for vertebrate Polycomb recruitment. This model is supported by analysis of DNA methyltransferase-deficient embryonic stem cells.

Tuning of ß-catenin activity is required to stabilize self-renewal of rat embryonic stem cells.

Stabilization of ß-catenin, through inhibition of glycogen synthase kinase 3 (GSK3) activity, in conjunction with inhibition of mitogen-activated protein kinase kinase 1/2 (MEK) promotes self-renewal of naïve-type mouse embryonic stem cells (ESC). In developmentally more advanced, primed-type, epiblast stem cells, however, ß-catenin activity induces differentiation. We investigated the response of rat ESCs to ß-catenin signaling and found that when maintained on feeder-support cells in the presence of a MEK inhibitor alone (1i culture), the derivation efficiency, growth, karyotypic stability, transcriptional profile, and differentiation potential of rat ESC cultures was similar to that of cell lines established using both MEK and GSK3 inhibitors (2i culture). Equivalent mouse ESCs, by comparison, differentiated in identical 1i conditions, consistent with insufficient ß-catenin activity. This interspecies difference in reliance on GSK3 inhibition corresponded with higher overall levels of ß-catenin activity in rat ESCs. Indeed, rat ESCs displayed widespread expression of the mesendoderm-associated ß-catenin targets, Brachyury and Cdx2 in 2i medium, and overt differentiation upon further increases in ß-catenin activity. In contrast, mouse ESCs were resistant to differentiation at similarly elevated doses of GSK3 inhibitor. Interestingly, without feeder support, moderate levels of GSK3 inhibition were necessary to support effective growth of rat ESC, confirming the conserved role for ß-catenin in ESC self-renewal. This work identifies ß-catenin signaling as a molecular rheostat in rat ESC, regulating self-renewal in a dose-dependent manner, and highlights the potential importance of controlling flux in this signaling pathway to achieve effective stabilization of naïve pluripotency.

Phenotypic characterisation of bovine alveolar macrophages reveals two major subsets with differential expression of CD163.

Bovine alveolar macrophages (AMs) defend the lungs against pathogens such as Mycobacterium bovis (M. bovis), the causative agent of bovine tuberculosis. However, little is known about the surface molecules expressed by bovine AMs and whether there is heterogeneity within the population. The purpose of this study was to characterise the bovine AM cell surface phenotype using flow cytometry. Bronchoalveolar lavage samples from four different calves were stained with a combination of antibodies against immune cell molecules prior to flow cytometric analysis. To assess the degree of expression, we considered the distribution and relative intensities of stained and unstained cells. We demonstrated that bovine AMs have high expression of CD172a, ADGRE1, CD206, and CD14, moderate expression of CD80, MHC II, CD1b, and CD40, low expression of CX3CR1 and CD86, and little or no expression of CD16 and CD26. Two distinct subsets of bovine AMs were identified based on CD163 expression. Subsequent analysis showed that the CD163+ subset had greater expression of other typical macrophage molecules compared to the CD163- subset, suggesting that these cells may perform different roles during infection. The characterisation of the uninfected bovine AM phenotype will provide a foundation for the examination of M. bovis-infected AMs.

MeCP2 binds to methylated DNA independently of phase separation and heterochromatin organisation.

Correlative evidence has suggested that the methyl-CpG-binding protein MeCP2 contributes to the formation of heterochromatin condensates via liquid-liquid phase separation. This interpretation has been reinforced by the observation that heterochromatin, DNA methylation and MeCP2 co-localise within prominent foci in mouse cells. The findings presented here revise this view. MeCP2 localisation is independent of heterochromatin as MeCP2 foci persist even when heterochromatin organisation is disrupted. Additionally, MeCP2 foci fail to show hallmarks of phase separation in live cells. Importantly, we find that mouse cellular models are highly atypical as MeCP2 distribution is diffuse in most mammalian species, including humans. Notably, MeCP2 foci are absent in Mus spretus which is a mouse subspecies lacking methylated satellite DNA repeats. We conclude that MeCP2 has no intrinsic tendency to form condensates and its localisation is independent of heterochromatin. Instead, the distribution of MeCP2 in the nucleus is primarily determined by global DNA methylation patterns.

Myocardial Infarction Across COVID-19 Pandemic Phases: Insights From the Veterans Health Affairs System.

Background Cardiovascular procedural treatments were deferred at scale during the COVID-19 pandemic, with unclear impact on patients presenting with non-ST-segment-elevation myocardial infarction (NSTEMI). Methods and Results In a retrospective cohort study of all patients diagnosed with NSTEMI in the US Veterans Affairs Healthcare System from January 1, 2019 to October 30, 2022 (n=67 125), procedural treatments and outcomes were compared between the prepandemic period and 6 unique pandemic phases: (1) acute phase, (2) community spread, (3) first peak, (4) post vaccine, (5) second peak, and (6) recovery. Multivariable regression analysis was performed to assess the association between pandemic phases and 30-day mortality. NSTEMI volumes dropped significantly with the pandemic onset (62.7% of prepandemic peak) and did not revert to prepandemic levels in subsequent phases, even after vaccine availability. Percutaneous coronary intervention and coronary artery bypass grafting volumes declined proportionally. Compared with the prepandemic period, patients with NSTEMI experienced higher 30-day mortality during Phases 2 and 3, even after adjustment for COVID-19-positive status, demographics, baseline comorbidities, and receipt of procedural treatment (adjusted odds ratio for Phases 2 and 3 combined, 1.26 [95% CI, 1.13-1.43], P<0.01). Patients receiving Veterans Affairs-paid community care had a higher adjusted risk of 30-day mortality compared with those at Veterans Affairs hospitals across all 6 pandemic phases. Conclusions Higher mortality after NSTEMI occurred during the initial spread and first peak of the pandemic but resolved before the second, higher peak-suggesting effective adaptation of care delivery but a costly delay to implementation. Investigation into the vulnerabilities of the early pandemic spread are vital to informing future resource-constrained practices.

Modulation of APLNR Signaling Is Required during the Development and Maintenance of the Hematopoietic System.

Apelin receptor (APLNR/AGTRLl1/APJ) marks a transient cell population during the differentiation of hematopoietic stem and progenitor cells (HSPCs) from pluripotent stem cells, but its function during the production and maintenance of hematopoietic stem cells is not clear. We generated an Aplnr-tdTomato reporter mouse embryonic stem cell (mESC) line and showed that HSPCs are generated exclusively from mesodermal cells that express Aplnr-tdTomato. HSPC production from mESCs was impaired when Aplnr was deleted, implying that this pathway is required for their production. To address the role of APLNR signaling in HSPC maintenance, we added APELIN ligands to ex vivo AGM cultures. Activation of the APLNR pathway in this system impaired the generation of long-term reconstituting HSPCs and appeared to drive myeloid differentiation. Our data suggest that the APLNR signaling is required for the generation of cells that give rise to HSCs, but that its subsequent downregulation is required for their maintenance.

A Stem Cell Reporter for Investigating Pluripotency and Self-Renewal in the Rat.

Rat embryonic stem cells (rESCs) are capable of contributing to all differentiated tissues, including the germ line in chimeric animals, and represent a unique, authentic alternative to mouse embryonic stem cells for studying stem cell pluripotency and self-renewal. Here, we describe an EGFP reporter transgene that tracks expression of the benchmark naive pluripotency marker gene Rex1 (Zfp42) in the rat. Insertion of the EGFP reporter gene downstream of the Rex1 promoter disrupted Rex1 expression, but REX1-deficient rESCs and rats were viable and apparently normal, validating this targeted knockin transgene as a neutral reporter. The Rex1-EGFP gene responded to self-renewal/differentiation factors and validated the critical role of ß-catenin/LEF1 signaling. The stem cell reporter also allowed the identification of functionally distinct sub-populations of cells within rESC cultures, thus demonstrating its utility in discriminating between cell states in rat stem cell cultures, as well as providing a tool for tracking Rex1 expression in the rat.

Signalling, cell cycle and pluripotency in embryonic stem cells.

Pluripotent mouse embryonic stem (ES) cells can be expanded in large numbers in vitro owing to a process of symmetrical self-renewal. Self-renewal entails proliferation with a concomitant suppression of differentiation. Here we describe how the cytokine leukaemia inhibitory factor (LIF) sustains self-renewal through activation of the transcription factor STAT3, and how two other signals - extracellular-signal-related kinase (ERK) and phosphatidylinositol-3-OH kinase (PI3K) - can influence differentiation and propagation, respectively. We relate these observations to the unusual cell-cycle properties of ES cells and speculate on the role of the cell cycle in maintaining pluripotency.

A novel approach to differentiate rat embryonic stem cells in vitro reveals a role for RNF12 in activation of X chromosome inactivation.

X chromosome inactivation (XCI) is a mammalian specific, developmentally regulated process relying on several mechanisms including antisense transcription, non-coding RNA-mediated silencing, and recruitment of chromatin remodeling complexes. In vitro modeling of XCI, through differentiation of embryonic stem cells (ESCs), provides a powerful tool to study the dynamics of XCI, overcoming the need for embryos, and facilitating genetic modification of key regulatory players. However, to date, robust initiation of XCI in vitro has been mostly limited to mouse pluripotent stem cells. Here, we adapted existing protocols to establish a novel monolayer differentiation protocol for rat ESCs to study XCI. We show that differentiating rat ESCs properly downregulate pluripotency factor genes, and present female specific Xist RNA accumulation and silencing of X-linked genes. We also demonstrate that RNF12 seems to be an important player in regulation of initiation of XCI in rat, acting as an Xist activator. Our work provides the basis to investigate the mechanisms directing the XCI process in a model organism different from the mouse.

Real-Time Sexing of Chicken Embryos and Compatibility with in ovo Protocols.

The chicken embryo is an established model system for studying early vertebrate development. One of the major advantages of this model is the facility to perform manipulations in ovo and then continue incubation and observe the effects on embryonic development. However, in common with other vertebrate models, there is a tendency to disregard the sex of the experimental chicken embryos, and this can lead to erroneous conclusions, a lack of reproducibility, and wasted efforts. That this neglect is untenable is emphasised by the recent demonstration that avian cells and tissues have an inherent sex identity and that male and female tissues respond differently to the same stimulus. These sexually dimorphic characteristics dictate that analyses and manipulations involving chicken embryos should always be performed using tissues/embryos of known sex. Current sexing protocols are unsuitable in many instances because of the time constraints imposed by most in ovo procedures. To address this lack, we have developed a real-time chicken sexing assay that is compatible with in ovo manipulations, reduces the number of embryos required, and conserves resources.

Reduced levels of dopamine and altered metabolism in brains of HPRT knock-out rats: a new rodent model of Lesch-Nyhan Disease.

Lesch-Nyhan disease (LND) is a severe neurological disorder caused by loss-of-function mutations in the gene encoding hypoxanthine phosphoribosyltransferase (HPRT), an enzyme required for efficient recycling of purine nucleotides. Although this biochemical defect reconfigures purine metabolism and leads to elevated levels of the breakdown product urea, it remains unclear exactly how loss of HPRT activity disrupts brain function. As the rat is the preferred rodent experimental model for studying neurobiology and diseases of the brain, we used genetically-modified embryonic stem cells to generate an HPRT knock-out rat. Male HPRT-deficient rats were viable, fertile and displayed normal caged behaviour. However, metabolomic analysis revealed changes in brain biochemistry consistent with disruption of purine recycling and nucleotide metabolism. Broader changes in brain biochemistry were also indicated by increased levels of the core metabolite citrate and reduced levels of lipids and fatty acids. Targeted MS/MS analysis identified reduced levels of dopamine in the brains of HPRT-deficient animals, consistent with deficits noted previously in human LND patients and HPRT knock-out mice. The HPRT-deficient rat therefore provides a new experimental platform for future investigation of how HPRT activity and disruption of purine metabolism affects neural function and behaviour.

Tuning differentiation signals for efficient propagation and in vitro validation of rat embryonic stem cell cultures.

The rat is one of the most commonly used laboratory animals in biomedical research and the recent isolation of genuine pluripotent rat embryonic stem (ES) cell lines has provided new opportunities for applying contemporary genetic engineering techniques to the rat and enhancing the use of this rodent in scientific research. Technical refinements that improve the stability of the rat ES cell cultures will undoubtedly further strengthen and broaden the use of these stem cells in biomedical research. Here, we describe a relatively simple and robust protocol that supports the propagation of germ line competent rat ES cells, and outline how tuning stem cell signaling using small molecule inhibitors can be used to both stabilize self-renewal of rat ES cell cultures and aid evaluation of their differentiation potential in vitro.

Ovine-induced pluripotent stem cells can contribute to chimeric lambs.

Pluripotential stem cells from livestock offer an exciting prospect for the biotechnology industry. Applying strategies established for the derivation of murine induced pluripotential stem cells (iPSCs), we have isolated ovine iPSCs that can give rise to cells characteristic of all three germ cell layers both in vitro from embryoid bodies and in teratomas in vivo. Furthermore, although at a low level, these ovine iPS cells can contribute to live-born chimeric lambs. Colonies derived from ovine embryonic fibroblasts transfected with murine cMyc, Klf4, Oct4, and Sox2 displayed smooth domes with sharp edges when grown in human embryonic stem cell (ESC) medium but not in mouse ESC medium. These ovine iPSCs were alkaline phosphatase positive, expressed Nanog, and had a normal karyotype. These cells represent an important step in the understanding of mechanistic nature of pluripotency in ungulates.

Reprogramming pig fetal fibroblasts reveals a functional LIF signaling pathway.

Distinct signaling pathways are reported to maintain pluripotency in embryo-derived stem cells. Mouse embryonic stem cells (ESCs) respond to leukemia inhibitory factor (LIF) and bone morphogenetic protein (BMP)-mediated activity, whereas human ESCs depend upon Fibroblast growth factor (FGF) and activin signaling. In the majority of mammals investigated, however, the signals that support stem cell pluripotency are not well defined, as is evident by the persistent difficulties in maintaining authentic stable ESC lines. Induction of pluripotency by transcription factor-mediated reprogramming could provide an alternative way to produce ESC-like cells from nonpermissive species, and facilitate identification of core ESC signaling requirements. To evaluate the effectiveness of this approach in pigs, we transduced porcine foetal fibroblasts with retroviruses expressing Oct4, Sox2, Klf4, and c-Myc, and maintained the resulting cultures in medium containing either LIF or FGF2. Alkaline phosphatase positive colonies with compact, mouse ESC-like morphology were preferentially recovered using serum-free medium supplemented with LIF. These cell lines expressed the endogenous stem cell transcription factors, OCT4, NANOG, and SOX2, and the cell surface marker SSEA-4, consistent with acquisition of an undifferentiated state. However, restricted differentiation potential, and persistent expression of retroviral transgenes indicated that reprogramming was incomplete. Interestingly, LIF activated both the transcription factor STAT3 and its target gene SOCS3, and stimulated cell growth, indicating functional coupling of the signaling pathway in these cells. This demonstration of LIF-dependence in reprogrammed pig cells supports the notion that the connection between LIF/STAT3 signaling and the core regulatory network of pluripotent stem cells is a conserved pathway in mammals.