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Background & aimsThe stimulator of interferon genes (STING)/TANK-binding kinase 1 (TBK1) pathway is vital in mediating innate immune and inflammatory responses during oxidative/endoplasmic reticulum (ER) stress. However, it remains unknown whether macrophage thioredoxin-interacting protein (TXNIP) may regulate TBK1 function and cell death pathways during oxidative/ER stress.MethodsA mouse model of hepatic ischaemia/reperfusion injury (IRI), the primary hepatocytes, and bone marrow-derived macrophages were used in the myeloid-specific TXNIP knockout (TXNIPM-KO) and TXNIP-proficient (TXNIPFL/FL) mice.ResultsThe TXNIPM-KO mice were resistant to ischaemia/reperfusion (IR) stress-induced liver damage with reduced serum alanine aminotransferase (ALT)/aspartate aminotransferase (AST) levels, macrophage/neutrophil infiltration, and pro-inflammatory mediators compared with the TXNIPFL/FL controls. IR stress increased TXNIP, p-STING, and p-TBK1 expression in ischaemic livers. However, TXNIPM-KO inhibited STING, TBK1, interferon regulatory factor 3 (IRF3), and NF-κB activation with interferon-β (IFN-β) expression. Interestingly, TXNIPM-KO augmented nuclear factor (erythroid-derived 2)-like 2 (NRF2) activity, increased antioxidant gene expression, and reduced macrophage reactive oxygen species (ROS) production and hepatic apoptosis/necroptosis in IR-stressed livers. Mechanistically, macrophage TXNIP deficiency promoted cylindromatosis (CYLD), which colocalised and interacted with NADPH oxidase 4 (NOX4) to enhance NRF2 activity by deubiquitinating NOX4. Disruption of macrophage NRF2 or its target gene 2',5' oligoadenylate synthetase-like 1 (OASL1) enhanced Ras GTPase-activating protein-binding protein 1 (G3BP1) and TBK1-mediated inflammatory response. Notably, macrophage OASL1 deficiency induced hepatocyte apoptotic peptidase activating factor 1 (APAF1), cytochrome c, and caspase-9 activation, leading to increased caspase-3-initiated apoptosis and receptor-interacting serine/threonine-protein kinase 3 (RIPK3)-mediated necroptosis.ConclusionsMacrophage TXNIP deficiency enhances CYLD activity and activates the NRF2-OASL1 signalling, controlling IR stress-induced liver injury. The target gene OASL1 regulated by NRF2 is crucial for modulating STING-mediated TBK1 activation and Apaf1/cytochrome c/caspase-9-triggered apoptotic/necroptotic cell death pathway. Our findings underscore a novel role of macrophage TXNIP-mediated CYLD-NRF2-OASL1 axis in stress-induced liver inflammation and cell death, implying the potential therapeutic targets in liver inflammatory diseases.Lay summaryLiver inflammation and injury induced by ischaemia and reperfusion (the absence of blood flow to the liver tissue followed by the resupply of blood) is a significant cause of hepatic dysfunction and failure following liver transplantation, resection, and haemorrhagic shock. Herein, we uncover an underlying mechanism that contributes to liver inflammation and cell death in this setting and could be a therapeutic target in stress-induced liver inflammatory injury.

The CRISPR/Cas9 system has unprecedentedly revolutionized genome-editing technology, which is being successfully applied virtually in all branches of biological sciences. Although much success has been attained in gene manipulation, still the majority of methods are laborious and non-integration-free, and require prolonged time for the expansion of mutant cell pools/clones, while fewer cells exhibit functional knockout efficiency. To overcome these obstacles, here, we describe an efficient, inexpensive, integration-free, and rapid one-step protocol for CRISPR/Cas9-assisted gene knockout in murine pluripotent stem cells (PSCs). Our protocol has streamlined both the liposome-based transfection system and screening strategy to work more efficiently with small numbers of PSCs (∼2.0 × 104 cells) and to minimize laborious steps of lentiviral packaging, transduction, and single-clone passaging. In our method, around 90% (CI = 95%, 79.5230%–100%) of PSC colonies harbored functional knockout in the context of protein expression. Therefore, the current protocol is technically feasible, time-saving, and highly efficient for genome editing in pluripotent stem cells.

Pluripotent stem cells (embryonic stem cells, ESCs, and induced pluripotent stem cells, iPSCs) have the capacity to generate neural progenitors that are intrinsically patterned to undergo differentiation into specific neuronal subtypes and express in vivo properties that match the ones formed during normal embryonic development. Remarkable progress has been made in this field during recent years thanks to the development of more refined protocols for the generation of transplantable neuronal progenitors from pluripotent stem cells, and the access to new tools for tracing of neuronal connectivity and assessment of integration and function of grafted neurons. Recent studies in brains of neonatal mice or rats, as well as in rodent models of brain or spinal cord damage, have shown that ESC- or iPSC-derived neural progenitors can be made to survive and differentiate after transplantation, and that they possess a remarkable capacity to extend axons over long distances and become functionally integrated into host neural circuitry. Here, we summarize these recent developments in the perspective of earlier studies using intracerebral and intraspinal transplants of primary neurons derived from fetal brain, with special focus on the ability of human ESC- and iPSC-derived progenitors to reconstruct damaged neural circuitry in cortex, hippocampus, the nigrostriatal system and the spinal cord, and we discuss the intrinsic and extrinsic factors that determine the growth properties of the grafted neurons and their capacity to establish target-specific long-distance axonal connections in the damaged host brain.

Mitochondrial diseases are heterogeneous disorders, caused by mitochondrial dysfunction. Mitochondria are not regulated solely by nuclear genomic DNA but by mitochondrial DNA. It is difficult to develop effective therapies for mitochondrial disease because of the lack of mitochondrial disease models. Mitochondrial myopathy, encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) is one of the major mitochondrial diseases. The aim of this study was to generate MELAS-specific induced pluripotent stem cells (iPSCs) and to demonstrate that MELAS-iPSCs can be models for mitochondrial disease. We successfully established iPSCs from the primary MELAS-fibroblasts carrying 77.7% of m.3243A>G heteroplasmy. MELAS-iPSC lines ranged from 3.6% to 99.4% of m.3243A>G heteroplasmy levels. The enzymatic activities of mitochondrial respiratory complexes indicated that MELAS-iPSC-derived fibroblasts with high heteroplasmy levels showed a deficiency of complex I activity but MELAS-iPSC-derived fibroblasts with low heteroplasmy levels showed normal complex I activity. Our data indicate that MELAS-iPSCs can be models for MELAS but we should carefully select MELAS-iPSCs with appropriate heteroplasmy levels and respiratory functions for mitochondrial disease modeling. [ABSTRACT FROM AUTHOR]

In Part I, we documented differences in cryopreservation success measured by membrane integrity in four mouse embryonic stem cell (mESC) lines from different genetic backgrounds (BALB/c, CBA, FVB, and 129R1), and we demonstrated a potential biophysical basis for these differences through a comparative study characterizing the membrane permeability characteristics and osmotic tolerance limits of each cell line. Here we use these values to predict optimal cryoprotectants, cooling rates, warming rates, and plunge temperatures. We subsequently verified these predictions experimentally for their effects on post-thaw recovery. From this study, we determined that a cryopreservation protocol utilizing 1M propylene glycol, a cooling rate of 1°C/minute, and plunging into liquid nitrogen at −41°C, combined with subsequent warming in a 22°C water bath with agitation, significantly improved post-thaw recovery for three of the four mESC lines, and did not diminish post-thaw recovery for our single exception. It is proposed that this protocol can be successfully applied to most mESC lines beyond those included within this study once the effect of propylene glycol on mESC gene expression, growth characteristics, and germ-line transmission has been determined. Mouse ESC lines with poor survival using current standard cryopreservation protocols or our proposed protocol can be optimized on a case-by-case basis using the method we have outlined over two papers. For our single exception, the CBA cell line, a cooling rate of 5°C/minute in the presence of 1.0M dimethyl sulfoxide or 1.0M propylene glycol, combined with plunge temperature of −80°C was optimal. [ABSTRACT FROM AUTHOR]

Abstract: The post-thaw recovery of mouse embryonic stem cells (mESCs) is often assumed to be adequate with current methods. However as this publication will show, this recovery of viable cells actually varies significantly by genetic background. Therefore there is a need to improve the efficiency and reduce the variability of current mESC cryopreservation methods. To address this need, we employed the principles of fundamental cryobiology to improve the cryopreservation protocol of four mESC lines from different genetic backgrounds (BALB/c, CBA, FVB, and 129R1 mESCs) through a comparative study characterizing the membrane permeability characteristics and membrane integrity osmotic tolerance limits of each cell line. In the companion paper, these values were used to predict optimal cryoprotectants, cooling rates, warming rates, and plunge temperatures, and then these predicted optimal protocols were validated against standard freezing protocols. [Copyright &y& Elsevier]

Abstract: Accumulation of mutations in embryonic stem (ES) cells would be detrimental to an embryo derived from these cells, and would adversely affect multiple organ systems and tissue types. ES cells have evolved multiple mechanisms to preserve genomic integrity that extend beyond those found in differentiated cell types. The present study queried whether mismatch repair (MMR) and base-excision repair (BER) may play a role in the maintenance of murine ES cell genomes. The MMR proteins Msh2 and Msh6 are highly elevated in mouse ES cells compared with mouse embryo fibroblasts (MEFs), as are Pms2 and Mlh1, albeit to a lesser extent. Cells transfected with an MMR reporter plasmid showed that MMR repair capacity is low in MEFs, but highly active in wildtype ES cells. As expected, an ES cell line defective in MMR was several-fold less effective in repair level than wildtype ES cells. Like proteins that participate in MMR, the level of proteins involved in BER was elevated in ES cells compared with MEFs. When BER activity was examined biochemically using a uracil-containing oligonucleotide template, repair activity was higher in ES cells compared with MEFs. The data are consistent with the suggestion that ES cells have multiple mechanisms, including highly active MMR and BER that preserve genetic integrity and minimize the accumulation of mutations. [Copyright &y& Elsevier]

The article presents a reprint of the article "Nuclear Transfer: An Example of Responsive Epistemologies," by Giuseppe Testa, which appeared in the 2006 issue of "Proceedings of the Max Planck Institute for the History of Science." It discusses several developments in mammalian cloning or nuclear transfer, and the ethical issues related with cloning and embryonic stem (ES) cell studies. It examines experimental practices and artefacts related to cloning, such as altered nuclear transfer (ANT).

Embryonic stem (ES) cells which constitutively express the Pdx-1, Ngn-3, NeuroD1, Nkx2.2, and Nkx6.1 transcription factors were engineered by means of lentiviral vectors, following a multi-step infection procedure to successively generate ES cell lines expressing one, two, and three factors, respectively. Each ES cell line was allowed to differentiate into nestin+/Isl-1+ endocrine precursors, then into more mature pancreatic cells, and subsequently analysed for expression of Glc, Ins, and Sst, markers of α, β and δ cells, respectively. Each ES cell line generated displayed a unique pattern of gene expression. The ES cell line expressing NeuroD1 displayed vastly elevated levels of Glc, Ins-1, Ins-2 and Sst, and showed an increase in Pdx-1, Pax-4, Nkx6.1, Isl-1, Glut-2 and GK transcript levels. Furthermore, immunofluorescence analysis revealed that differentiation of NeuroD1-expressing ES cells in nestin+/Isl-1+ multilineage progenitors, followed by the formation of C-peptide+/insulin+ clusters, was accelerated. Together, these results indicate that stable expression of NeuroD1 in ES cells facilitates differentiation into endocrine and insulin-producing cells. [ABSTRACT FROM AUTHOR]

Biologists rely on morphology, function and specific markers to define the differentiation status of cells. Transcript profiling has expanded the repertoire of these markers by providing the snapshot of cellular status that reflects the activity of all genes. However, such data have been used only to assess relative similarities and differences of these cells. Here we show that principal component analysis of global gene expression profiles map cells in multidimensional transcript profile space and the positions of differentiating cells progress in a stepwise manner along trajectories starting from undifferentiated embryonic stem (ES) cells located in the apex. We present three ‘cell lineage trajectories’, which represent the differentiation of ES cells into the first three lineages in mammalian development: primitive endoderm, trophoblast and primitive ectoderm/neural ectoderm. The positions of the cells along these trajectories seem to reflect the developmental potency of cells and can be used as a scale for the potential of cells. Indeed, we show that embryonic germ cells and induced pluripotent cells are mapped near the origin of the trajectories, whereas mouse embryo fibroblast and fibroblast cell lines are mapped near the far end of the trajectories. We suggest that this method can be used as the non-operational semi-quantitative definition of cell differentiation status and developmental potency. Furthermore, the global expression profiles of cell lineages provide a framework for the future study of in vitro and in vivo cell differentiation. [ABSTRACT FROM PUBLISHER]

Embryonic stem (ES) cells represent a unique cellular model to recapitulate in vitro early steps of embryonic development and an unlimited cellular source in therapy for many diseases, as well as targets for drug discovery and toxicology screens. Although previous studies have reported epidermal differentiation of mouse and human embryonic stem (huES) cells, the heterogeneity of the resulting cell culture impairs the evaluation of differentiated cells for cell therapy. We report here the reproducible isolation of a homogenous ectodermal cell population, IT1, from human ES cells. Like primary cells, IT1 cells remain homogenous over 15 passages, expand up to 60 population doublings, and then die through senescence. Accordingly, IT1 cells display a normal karyotype and a somatic cell cycle kinetics and do not produce teratoma in nude mice. The production of K14-expressing epithelial cells driven by p63 expression strengthens the ectodermal nature of IT1 cells. Since IT1 can be isolated from different huES cell lines, it may provide a ready source of ectodermal progenitors for the development of a toxicology cell model, new-drug-screening strategies, and cell therapy transplantation. [ABSTRACT FROM AUTHOR]

Mouse embryonic stem (ES) cells have the pluripotent ability to differentiate in vitro into various cell lineages, including neurons. Adding chick dorsal root ganglion (DRG) conditioned medium (CM) to the culture medium promotes the differentiation of ES cells into neurons. We determined the types of neurons that differentiate from ES cells. The addition of DRG-CM caused nearly half of all ES cells on the periphery of the colony sphere to differentiate into neurons. Immunofluorescence analysis showed that the neurons that differentiated from ES cells were mainly motor, GABAergic, serotonergic, and cholinergic neurons. Of particular note, flow cytometry showed that approximately 50% of βIII-tubulin-positive neurons were motor neurons. This indicates that DRG-CM induces ES cells to differentiate into motor neurons as target of DRG neurons (sensory neurons). [Copyright &y& Elsevier]

The article examines whether differentiation potential of parthenogenetic embryonic stem cells (pES) is improved by nuclear transfer (NT). Several second- and third-generation NT-pES cell lines were established from original pES cell by NT. These were examined for their potential to differential in vivo and in vitro.

Therapeutic cloning, whereby nuclear transfer (NT) is used to generate embryonic stem cells (ESCs) from blastocysts, has been demonstrated successfully in mice and cattle. However, if NT-ESCs have abnormalities, such as those associated with the offspring produced by reproductive cloning, their scientific and medical utilities might prove limited. To evaluate the characteristics of NT-ESCs, we established more than 150 NT-ESC lines from adult somatic cells of several mouse strains. Here, we show that these NT-ESCs were able to differentiate into all functional embryonic tissues in vivo. Moreover, they were identical to blastocystderived ESCs in terms of their expression of pluripotency markers in the presence of tissue-dependent differentially DNA methylated regions, in DNA microarray profiles, and in high-coverage gene expression profiling. Importantly, the NT procedure did not cause irreversible damage to the nuclei. These similarities of NT-ESCs and ESCs indicate that murine therapeutic cloning by somatic cell NT can provide a reliable model for preclinical stem cell research. [ABSTRACT FROM AUTHOR]

The in vitro differentiation of embryonic stem (ES) cells into specific phenotypes plays an important role in the development of stem cell therapy, tissue engineering and regenerative medicine. Currently, there are no biological assays able to characterise and monitor in situ and in real-time ES cells during the differentiation process. We applied Raman micro-spectroscopy to compare undifferentiated murine ES cells with murine ES cells in the differentiation process. The most significant differences between undifferentiated and differentiated ES cells (16 days differentiation via formation of embryoid body) were related to the nucleic acids. The decrease in the magnitude of RNA 813 cm-1 Raman peaks (25%) in the differentiated ES cells in comparison to undifferentiated ES cells, suggests that part of the RNA in the ES cells is used for the synthesis of specific proteins in the early stages of differentiation. In the same time, the DNA 786 cm-1 Raman peaks were lower by 50%, indicating that the differentiated cells are more in the G1 phase than S, G2 or M phases of the cell cycle. This result suggests that the proliferation rate of differentiated cells is reduced following development of a mature phenotype. This study shows the feasibility of using Raman micro-spectroscopy to monitor in situ and in real-time the differentiation of ES cells by using the intensity of Raman peak of nucleic acids as differentiation markers. [Copyright &y& Elsevier]

The inability of adult cardiomyocytes to divide to a significant extent and regenerate the myocardium after injury leads to permanent deficits in the number of functional cells, which can contribute to the development and progression of heart failure. The transplantation of skeletal myoblasts or stem cells or cardiomyocytes derived from them into the injured myocardium is a novel and promising approach in the treatment of cardiac disease and the restoration of myocardial function. In this article, skeletal myoblasts and embryonic and bone marrow stem cells are discussed in the context of their potential therapeutic use in cardiac failure. The state of the art in both laboratory and clinic is presented. We discuss current and intrinsic limitations of cardiac cellular transplantation and suggest directions for future research. [Copyright &y& Elsevier]