Activated Natural Killer Cell Inoculation Alleviates Fibrotic Liver Pathology in a Carbon Tetrachloride-Induced Liver Cirrhosis Mouse Model.

Activated hepatic stellate cells (HSCs) play a detrimental role in liver fibrosis progression. Natural killer (NK) cells are known to selectively recognize abnormal or transformed cells via their receptor activation and induce target cell apoptosis and, therefore, can be used as a potential therapeutic strategy for liver cirrhosis. In this study, we examined the therapeutic effects of NK cells in the carbon tetrachloride (CCl4)-induced liver cirrhosis mouse model. NK cells were isolated from the mouse spleen and expanded in the cytokine-stimulated culture medium. Natural killer group 2, member D (NKG2D)-positive NK cells were significantly increased after a week of expansion in culture. The intravenous injection of NK cells significantly alleviated liver cirrhosis by reducing collagen deposition, HSC marker activation, and macrophage infiltration. For in vivo imaging, NK cells were isolated from codon-optimized luciferase-expressing transgenic mice. Luciferase-expressing NK cells were expanded, activated and administrated to the mouse model to track them. Bioluminescence images showed increased accumulation of the intravenously inoculated NK cells in the cirrhotic liver of the recipient mouse. In addition, we conducted QuantSeq 3' mRNA sequencing-based transcriptomic analysis. From the transcriptomic analysis, 33 downregulated genes in the extracellular matrix (ECM) and 41 downregulated genes involved in the inflammatory response were observed in the NK cell-treated cirrhotic liver tissues from the 1532 differentially expressed genes (DEGs). This result indicated that the repetitive administration of NK cells alleviated the pathology of liver fibrosis in the CCl4-induced liver cirrhosis mouse model via anti-fibrotic and anti-inflammatory mechanisms. Taken together, our research demonstrated that NK cells could have therapeutic effects in a CCl4-induced liver cirrhosis mouse model. In particular, it was elucidated that extracellular matrix genes and inflammatory response genes, which were mainly affected after NK cell treatment, could be potential targets.

iPSCs in NK Cell Manufacturing and NKEV Development.

Natural killer (NK) cell immunotherapies for cancer can complement existing T cell therapies while benefiting from advancements already made in the immunotherapy field. For NK cell manufacturing, induced pluripotent stem cells (iPSCs) offer advantages including eliminating donor variation and providing an ideal platform for genome engineering. At the same time, extracellular vesicles (EVs) have become a major research interest, and purified NK cell extracellular vesicles (NKEVs) have been shown to reproduce the key functions of their parent NK cells. NKEVs have the potential to be developed into a standalone therapeutic with reduced complexity and immunogenicity compared to cell therapies. This review explores the role iPSC technology can play in both NK cell manufacturing and NKEV development.

iPSC-Derived Natural Killer Cells for Cancer Immunotherapy.

The discovery of human pluripotent stem cells (PSCs) at the turn of the century opened the door to a new generation of regenerative medicine research. Among PSCs, the donors available for induced pluripotent stem cells (iPSCs) are greatest, providing a potentially universal cell source for all types of cell therapies including cancer immunotherapies using natural killer (NK cells). Unlike primary NK cells, those prepared from iPSCs can be prepared with a homogeneous quality and are easily modified to exert a desired response to tumor cells. There already exist several protocols to genetically modify and differentiate iPSCs into NK cells, and each has its own advantages with regards to immunotherapies. In this short review, we detail the benefits of using iPSCs in NK cell immunotherapies and discuss the challenges that must be overcome before this approach becomes mainstream in the clinic.

Effect of Multiple Reflows on the Interfacial Reactions and Mechanical Properties of an Sn-0.5Cu-Al(Si) Solder and a Cu Substrate.

In this study, the interfacial reactions and mechanical properties of solder joints after multiple reflows were observed to evaluate the applicability of the developed materials for high-temperature soldering for automotive electronic components. The microstructural changes and mechanical properties of Sn-Cu solders regarding Al(Si) addition and the number of reflows were investigated to determine their reliability under high heat and strong vibrations. Using differential scanning calorimetry, the melting points were measured to be approximately 227, 230, and 231 °C for the SC07 solder, SC-0.01Al(Si), and SC-0.03Al(Si), respectively. The cross-sectional analysis results showed that the total intermetallic compounds (IMCs) of the SC-0.03Al(Si) solder grew the least after the as-reflow, as well as after 10 reflows. Electron probe microanalysis and transmission electron microscopy revealed that the Al-Cu and Cu-Al-Sn IMCs were present inside the solders, and their amounts increased with increasing Al(Si) content. In addition, the Cu6Sn5 IMCs inside the solder became more finely distributed with increasing Al(Si) content. The Sn-0.5Cu-0.03Al(Si) solder exhibited the highest shear strength at the beginning and after 10 reflows, and ductile fracturing was observed in all three solders. This study will facilitate the future application of lead-free solders, such as an Sn-Cu-Al(Si) solder, in automotive electrical components.

Minimally Invasive Plate Osteosynthesis (MIPO) technique for complex tibial shaft fracture.

TTo evaluate the clinical and radiological results of the treatment of complex tibial shaft fracture (AO/OTA type 42-C) with minimally invasive plate osteosynthesis(MIPO). Twenty patients diagnosed with complex tibial shaft fracture without extension to the articular surface and treated with MIPO, including 9 cases of AO/OTA type 42-C2 and 11 cases of AO/OTA type 42-C3, 6 of which were open fractures. External fixation was used for open fractures until the soft tissue damage had healed; then, 2nd stage operation with MIPO was performed to stabilize the fracture. Each patient was followed up for a minimum of 12 months. The mean time to union was 20.1 weeks. Delayed union was observed in 4 cases. Angular deformity, length shortening and non-union were not observed. Severely comminuted and open fractures of the tibial shaft may benefit from temporary external fixation prior to performing MIPO.

OVOL1 Influences the Determination and Expansion of iPSC Reprogramming Intermediates.

During somatic cell reprogramming to induced pluripotent stem cells (iPSCs), fibroblasts undergo dynamic molecular changes, including a mesenchymal-to-epithelial transition (MET) and gain of pluripotency; processes that are influenced by Yamanaka factor stoichiometry. For example, in early reprogramming, high KLF4 levels are correlated with the induction of functionally undefined, transiently expressed MET genes. Here, we identified the cell-surface protein TROP2 as a marker for cells with transient MET induction in the high-KLF4 condition. We observed the emergence of cells expressing the pluripotency marker SSEA-1+ mainly from within the TROP2+ fraction. Using TROP2 as a marker in CRISPR/Cas9-mediated candidate screening of MET genes, we identified the transcription factor OVOL1 as a potential regulator of an alternative epithelial cell fate characterized by the expression of non-iPSC MET genes and low cell proliferation. Our study sheds light on how reprogramming factor stoichiometry alters the spectrum of intermediate cell fates, ultimately influencing reprogramming outcomes.

Microhomology-assisted scarless genome editing in human iPSCs.

Gene-edited induced pluripotent stem cells (iPSCs) provide relevant isogenic human disease models in patient-specific or healthy genetic backgrounds. Towards this end, gene targeting using antibiotic selection along with engineered point mutations remains a reliable method to enrich edited cells. Nevertheless, integrated selection markers obstruct scarless transgene-free gene editing. Here, we present a method for scarless selection marker excision using engineered microhomology-mediated end joining (MMEJ). By overlapping the homology arms of standard donor vectors, short tandem microhomologies are generated flanking the selection marker. Unique CRISPR-Cas9 protospacer sequences nested between the selection marker and engineered microhomologies are cleaved after gene targeting, engaging MMEJ and scarless excision. Moreover, when point mutations are positioned unilaterally within engineered microhomologies, both mutant and normal isogenic clones are derived simultaneously. The utility and fidelity of our method is demonstrated in human iPSCs by editing the X-linked HPRT1 locus and biallelic modification of the autosomal APRT locus, eliciting disease-relevant metabolic phenotypes.

Probabilistic evaluation of the material properties of the in vivo subject-specific articular surface using a computational model.

This article used probabilistic analysis to evaluate material properties of the in vivo subject-specific tibiofemoral (TF) joint model. Sensitivity analysis, based on a Monte Carlo (MC) method, was performed using a subject-specific finite element (FE) model generated from in vivo computed tomography (CT) and magnetic resonance imaging (MRI) data, subjected to two different loading conditions. Specifically, the effects of inherent uncertainty in ligament stiffness, horn attachment stiffness, and articular surface material properties were assessed using multifactorial global sensitivity analysis. The MRI images were taken before and after axial compression, and when the flexion condition had been maintained at up to 90 degree flexion in the subject-specific knee joint. The loading conditions of the probabilistic subject-specific FE model (axial compression and 90 degree flexion) were similar to the MRI acquisition setup. We were able to detect the influence of material parameters while maintaining the potential effect of parametric interactions. Throughout the in silico property optimization, a subject-specific FE model was used and less sensitive parameters were eliminated in the global sensitivity method. Soft tissue material properties were estimated using an optimization procedure that involved the minimization of the differences between the kinematics predicted by the subject-specific model and those obtained through in vivo subject-specific data. The results of this approach suggest that the articular surface mechanical properties could be found by using in vivo measurements, which clarifies the valuable tool for future subject-specific studies related to TF joint scaffolds, allografts and biologics. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 1390-1400, 2017.

Engineering the AAVS1 locus for consistent and scalable transgene expression in human iPSCs and their differentiated derivatives.

The potential use of induced pluripotent stem cells (iPSCs) in personalized regenerative medicine applications may be augmented by transgenics, including the expression of constitutive cell labels, differentiation reporters, or modulators of disease phenotypes. Thus, there is precedence for reproducible transgene expression amongst iPSC sub-clones with isogenic or diverse genetic backgrounds. Using virus or transposon vectors, transgene integration sites and copy numbers are difficult to control, and nearly impossible to reproduce across multiple cell lines. Moreover, randomly integrated transgenes are often subject to pleiotropic position effects as a consequence of epigenetic changes inherent in differentiation, undermining applications in iPSCs. To address this, we have adapted popular TALEN and CRISPR/Cas9 nuclease technologies in order to introduce transgenes into pre-defined loci and overcome random position effects. AAVS1 is an exemplary locus within the PPP1R12C gene that permits robust expression of CAG promoter-driven transgenes. Gene targeting controls transgene copy number such that reporter expression patterns are reproducible and scalable by ∼2-fold. Furthermore, gene expression is maintained during long-term human iPSC culture and in vitro differentiation along multiple lineages. Here, we outline our AAVS1 targeting protocol using standardized donor vectors and construction methods, as well as provide practical considerations for iPSC culture, drug selection, and genotyping.

Inducible Transgene Expression in Human iPS Cells Using Versatile All-in-One piggyBac Transposons.

Transgenics is a mainstay of functional genomics. Conditionally overexpressing genes of interest (GOIs) helps to reveal their roles in the control of complex biological processes. Complemented by findings in classic animal model systems, recent advances in human embryonic stem cell (hESC) and patient-specific induced pluripotent stem cell (hiPSC) differentiation have led to sophisticated in vitro models of human development and disease. Yet, as transgenic elements encoding inducible systems must be introduced de novo into each genetically unique human stem cell line, robust and straightforward solutions to gene delivery are required. Transposons are a family of mobile DNA elements that have been adapted as experimental tools for stable genomic integration of transgenes. The piggyBac (PB) transposon from Trichoplusia ni presents a number of benefits over classic viral or BAC transgenesis: ease of application, simple integration-site mapping, and the unique capacity for traceless excision. Moreover, their large capacity permits the consolidation of multiple transgene components in a single vector system. In this chapter, we outline the features of a panel of "All-in-One" PB transposons designed for drug-inducible gene expression and provide guidelines to establish and validate populations or clones of transgenic hiPSCs.

The piggyBac Transposon as a Platform Technology for Somatic Cell Reprogramming Studies in Mouse.

Somatic cell reprogramming to induced pluripotent stem cells (iPSCs) is a revolutionary technology, with repercussions affecting modern functional genomics and regenerative medicine. Still, relatively little is known about the processes underlying this dramatic cellular and molecular metamorphosis. Reprogramming technology based on the implementation of piggyBac (PB) transposons has enabled studies of iPSC reprogramming mechanisms, shedding an increasing light on these processes. Unique characteristics of PB transposons such as efficient genomic integration, unlimited cargo capacity, robust gene expression, and even seamless excision highlight the importance of this transgenic tool in advancing stem cell biology. In this chapter, we provide a detailed overview of versatile primary iPSC generation from mouse somatic cells using PB transposons, and the subsequent establishment of robust secondary reprogramming systems. These protocols are highlighted with examples from recent studies as to how PB has been, and continues to be, conducive to the dissection of reprogramming processes at the cellular and molecular levels.

Reprogramming Roadblocks Are System Dependent.

Since the first generation of induced pluripotent stem cells (iPSCs), several reprogramming systems have been used to study its molecular mechanisms. However, the system of choice largely affects the reprogramming efficiency, influencing our view on the mechanisms. Here, we demonstrate that reprogramming triggered by less efficient polycistronic reprogramming cassettes not only highlights mesenchymal-to-epithelial transition (MET) as a roadblock but also faces more severe difficulties to attain a pluripotent state even post-MET. In contrast, more efficient cassettes can reprogram both wild-type and Nanog(-/-) fibroblasts with comparable efficiencies, routes, and kinetics, unlike the less efficient reprogramming systems. Moreover, we attribute a previously reported variation in the N terminus of KLF4 as a dominant factor underlying these critical differences. Our data establish that some reprogramming roadblocks are system dependent, highlighting the need to pursue mechanistic studies with close attention to the systems to better understand reprogramming.

KLF4 N-terminal variance modulates induced reprogramming to pluripotency.

As the quintessential reprogramming model, OCT3/4, SOX2, KLF4, and c-MYC re-wire somatic cells to achieve induced pluripotency. Yet, subtle differences in methodology confound comparative studies of reprogramming mechanisms. Employing transposons, we systematically assessed cellular and molecular hallmarks of mouse somatic cell reprogramming by various polycistronic cassettes. Reprogramming responses varied in the extent of initiation and stabilization of transgene-independent pluripotency. Notably, the cassettes employed one of two KLF4 variants, differing only by nine N-terminal amino acids, which generated dissimilar protein stoichiometry. Extending the shorter variant by nine N-terminal amino acids or augmenting stoichiometry by KLF4 supplementation rescued both protein levels and phenotypic disparities, implicating a threshold in determining reprogramming outcomes. Strikingly, global gene expression patterns elicited by published polycistronic cassettes diverged according to each KLF4 variant. Our data expose a Klf4 reference cDNA variation that alters polycistronic factor stoichiometry, predicts reprogramming hallmarks, and guides comparison of compatible public data sets.

Effects of Therapeutic Hypothermia on Apoptosis and Autophagy After Spinal Cord Injury in Rats.

STUDY DESIGN: Animal study. OBJECTIVE: To further investigate the effects of therapeutic hypothermia (TH), the present study compared autophagy and apoptosis after treatment with either therapeutic moderate systemic hypothermia or methylprednisolone sodium succinate (MP) in a rat model of acute spinal cord injury (SCI). SUMMARY OF BACKGROUND DATA: The neuroprotective effects of TH have recently become an important topic in the field of SCI research. METHODS: All rats were subjected to a 25-g/cm spinal cord contusion over the ninth thoracic vertebrae. After the induction of SCI, the control group did not receive any further treatment, TH group immediately received moderate systemic hypothermia for 4 hours, and MP group was administered high-dose MP. The rats were killed either 2 or 7 days after SCI, and the injured spinal cord tissues were obtained. Apoptosis and autophagy were assessed by immunohistochemical analyses and Western blot analyses. In addition, the microarchitecture of the autophagosomes was evaluated using transmission electron microscopy, and the motor activity of the rats was assessed using the Basso-Beattie-Bresnahan (BBB) locomotor rating scale. RESULTS: Compared with controls, there was a significant reduction in the expression levels of cleaved caspase-8, -9, and -3 in the TH- and MP-treated groups 2 days after SCI. Moreover, compared with the control group, the expression of LC3II and Beclin-1 exhibited a significant decrease on day 2 after treatment with TH. The numbers of transferase dUTP nicked-end labeling and LC3-positive cells were significantly lower on days 2 and 7. The Basso-Beattie-Bresnahan ratings were significantly higher 6 weeks after SCI in both the TH- and MP-treated groups than in the control group. CONCLUSION: Both TH and MP have neuroprotective effects on injured spinal cord tissues via the inhibition of apoptosis and autophagy. Thus, the application of moderate systemic hypothermia may be a useful treatment modality after acute SCI. LEVEL OF EVIDENCE: N/A.

Fluorescence turn-on response of a conjugated polyelectrolyte with intramolecular stack structure to biomacromolecules.

An anionic conjugated polyelectrolyte based on polydiphenylacetylene showed a significant fluorescence turn-on response to positively-charged proteins through a conformational relaxation of its intramolecular stack structure.

BRG1 directly regulates nucleosome structure and chromatin looping of the alpha globin locus to activate transcription.

Alpha globin expression must be regulated properly to prevent the occurrence of alpha-thalassemias, yet many questions remain unanswered regarding the mechanism of transcriptional activation. Identifying factors that regulate chromatin structure of the endogenous alpha globin locus in developing erythroblasts will provide important mechanistic insight. Here, we demonstrate that the BRG1 catalytic subunit of SWI/SNF-related complexes co-immunoprecipitates with GATA-1 and EKLF in murine fetal liver cells in vivo and is recruited to the far-upstream major-regulatory element (MRE) and alpha2 promoter. Furthermore, based on our analysis of Brg1(null/ENU1) mutant mice, BRG1 regulates DNase I sensitivity, H3ac, and H3K4me2 but not CpG methylation at both sites. Most importantly, BRG1 is required for chromatin loop formation between the MRE and alpha2 promoter and for maximal RNA Polymerase II occupancy at the alpha2 promoter. Consequently, Brg1 mutants express alpha globin mRNA at only 5-10% of wild-type levels and die at mid-gestation. These data identify BRG1 as a chromatin-modifying factor required for nucleosome remodeling and transcriptional activation of the alpha globin locus. These data also demonstrate that chromatin looping between the MRE and alpha2 promoter is required as part of the transcriptional activation mechanism.

C3 promotes expansion of CD8+ and CD4+ T cells in a Listeria monocytogenes infection.

It is known that C3 is required for optimal expansion of T cells during acute viral infections. However, it is not yet determined whether T cell responses to intracellular bacterial infections require C3. Therefore, we have investigated the requirement for C3 to elicit potent T cell responses to Listeria monocytogenes (LM). We show that expansion of Ag-specific CD8 and CD4 T cells during a primary response to LM was markedly reduced in the absence of C3 activity. Further studies indicated that, unlike in an influenza virus infection, the regulation of LM-specific T cell responses by C3 might not involve the downstream effector C5a. Moreover, reduced T cell responses to LM was not linked to defective maturation of dendritic cells or developmental anomalies in the peripheral T cell compartment of C3-deficient mice. Experiments involving adoptive transfer of C3-deficient CD8 T cells into the C3-sufficient environment of wild-type mice showed that these T cells do not have intrinsic proliferative defects, and a paracrine source of C3 will suffice for clonal expansion of CD8 T cells in vivo. However, stimulation of purified C3-deficient CD8 T cells by plastic-immobilized anti-CD3 showed that C3 promotes T cell proliferation directly, independent of its effects on APC. On the basis of these findings, we propose that diminished T cell responses to LM in C3-deficient mice might be at least in part due to lack of direct effects of C3 on T cells. These studies have furthered our understanding of C3-mediated regulation of T cell immunity to intracellular pathogens.

BRG1 requirement for long-range interaction of a locus control region with a downstream promoter.

The dynamic packaging of DNA into chromatin is a fundamental step in the control of diverse nuclear processes. Whereas certain transcription factors and chromosomal components promote the formation of higher-order chromatin loops, the co-regulator machinery mediating loop assembly and disassembly is unknown. Using mice bearing a hypomorphic allele of the BRG1 chromatin remodeler, we demonstrate that the Brg1 mutation abrogated a cell type-specific loop between the beta-globin locus control region and the downstream beta major promoter, despite trans-acting factor occupancy at both sites. By contrast, distinct loops were insensitive to the Brg1 mutation. Molecular analysis with a conditional allele of GATA-1, a key regulator of hematopoiesis, in a novel cell-based system provided additional evidence that BRG1 functions early in chromatin domain activation to mediate looping. Although the paradigm in which chromatin remodelers induce nucleosome structural transitions is well established, our results demonstrating an essential role of BRG1 in the genesis of specific chromatin loops expands the repertoire of their functions.

Controlling hematopoiesis through sumoylation-dependent regulation of a GATA factor.

GATA factors establish transcriptional networks that control fundamental developmental processes. Whereas the regulator of hematopoiesis GATA-1 is subject to multiple posttranslational modifications, how these modifications influence GATA-1 function at endogenous loci is unknown. We demonstrate that sumoylation of GATA-1 K137 promotes transcriptional activation only at target genes requiring the coregulator Friend of GATA-1 (FOG-1). A mutation of GATA-1 V205G that disrupts FOG-1 binding and K137 mutations yielded similar phenotypes, although sumoylation was FOG-1 independent, and FOG-1 binding did not require sumoylation. Both mutations dysregulated GATA-1 chromatin occupancy at select sites, FOG-1-dependent gene expression, and were rescued by tethering SUMO-1. While FOG-1- and SUMO-1-dependent genes migrated away from the nuclear periphery upon erythroid maturation, FOG-1- and SUMO-1-independent genes persisted at the periphery. These results illustrate a mechanism that controls trans-acting factor function in a locus-specific manner, and differentially regulated members of the target gene ensemble reside in distinct subnuclear compartments.

Dissecting molecular steps in chromatin domain activation during hematopoietic differentiation.

GATA factors orchestrate hematopoiesis via multistep transcriptional mechanisms, but the interrelationships and importance of individual steps are poorly understood. Using complementation analysis with GATA-1-null cells and mice containing a hypomorphic allele of the chromatin remodeler BRG1, we dissected the pathway from GATA-1 binding to cofactor recruitment, chromatin loop formation, and transcriptional activation. Analysis of GATA-1-mediated activation of the beta-globin locus, in which GATA-1 assembles dispersed complexes at the promoters and the distal locus control region (LCR), revealed molecular intermediates, including GATA-1-independent and GATA-1-containing LCR subcomplexes, both defective in promoting loop formation. An additional intermediate consisted of an apparently normal LCR complex and a promoter complex with reduced levels of total RNA polymerase II (Pol II) and Pol II phosphorylated at serine 5 of the carboxy-terminal domain. Reduced BRG1 activity solely compromised Pol II and serine 5-phosphorylated Pol II occupancy at the promoter, phenocopying the LCR-deleted mouse. These studies defined a hierarchical order of GATA-1-triggered events at a complex locus and establish a novel mechanism of long-range gene regulation.